Plant cells and plants which synthesize a starch with an increased final viscosity

ABSTRACT

The present invention relates to a plant cell which is genetically modified, the genetic modification leading to the reduction of the activity of one or more SSIII proteins which occur endogenously in the plant cell and to the reduction of the activity of one or more BEI proteins which occur endogenously in the plant cell and to the reduction of the activity of one or more BEII proteins which occur endogenously in the plant cell in comparison with corresponding plant cells, of wild-type plants, which have not been genetically modified. Further aspects of the invention relate to plants containing such plant cells, to a method for generating the plant cells and plants, and to the starch obtainable from them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/539,723, filed Jun. 20, 2005, which is the U.S. National StageApplication of International Application PCT/EP/03/014840, filed Dec.19, 2003, which claims benefit of EP 02028530.0, filed Dec. 19, 2002,and EP 03090275.3, filed Aug. 29, 2003.

DESCRIPTION

The present invention relates to plant cells and plants which aregenetically modified, the genetic modification leading to the reductionof the activity of SSIII and BEI and BEII proteins in comparison withcorresponding plant cells, of wild-type plants, which have not beengenetically modified. Furthermore, the present invention relates tomeans and methods for the generation of such plant cells and plants.Such plant cells and plants synthesize a modified starch which ischaracterized in that it has an amylose content of at least 30% and aphosphate content which is increased in comparison with starch fromcorresponding wild-type plants which have not been genetically modifiedand which have a final viscosity in the RVA analysis which is increasedover the prior art and/or a modified side-chain distribution and/or anincreased gel strength in the Texture Analyser and/or a modified granulemorphology and/or a modified mean granule size. The present inventionthus also relates to the starch synthesized by the plant cells andplants according to the invention, and to methods for producing thisstarch.

In view of the increasing importance which is currently attached toplant constituents as renewable raw materials, one of the tasks ofbiotechnology research is to attempt an adaptation of these vegetableraw materials to the requirements of the processing industry. In orderto make possible the use of renewable raw materials in as many fields ofapplication as possible, it is additionally necessary to arrive at verydiverse substances.

The polysaccharide starch is a polymer of chemically uniform units, theglucose molecules. However, it takes the form of a highly complexmixture of different forms of molecules which differ with regard totheir degree of polymerization and the occurrence of branches of theglucose chains. Starch is therefore no uniform raw material. Onedistinguishes between two chemically different components of starch,amylose and amylopectin. In typical plants used for starch productionsuch as, for example, maize, wheat or potato, amylose starch accountsfor approximately 20%-30% and amylopectin starch for approximately70%-80% of the starch synthesized. Amylose has long been regarded as alinear polymer consisting of α-1,4-glycosidically linked α-D-glucosemonomers. However, more recent studies have demonstrated the presence ofα-1,6-glycosidic branch points (approx. 0.1%) (Hizukuri and Takagi,Carbohydr. Res. 134, (1984), 1-10; Takeda et al., Carbohydr. Res. 132,(1984), 83-92).

Various methods are available for determining the amylose content. Someof these methods are based on the iodine-binding capacity of amylose,which can determine potentiometrically (Banks & Greenwood, in W. Banks &C. T. Greenwood, Starch and its components (pp. 51-66), Edinburgh,Edinburgh University Press), amperometrically (Larson et al., AnalyticalChemistry 25(5), (1953), 802-804) or spectrophotometrically (Morrison &Laignelet, J. Cereal Sc. 1, (1983), 9-20). The amylose content may alsobe determined calorimetrically by means of DSC (differential scanningcalorimetry) measurements (Kugimiya & Donovan, Journal of Food Science46, (1981), 765-770; Sievert & Holm, Starch/Stärke 45 (4), (1993),136-139). It is furthermore possible to determine the amylose contentvia the use of SEC (size exclusion chromatography) chromatography ofnative or debranched starch. This method has been recommended inparticular for determining the amylose content of genetically modifiedstarches (Gérard et al., Carbohydrate Polymers 44, (2001), 19-27).

In contrast to amylose, amylopectin shows a higher degree of branchingand has approximately 4% of branch points brought about by theoccurrence of additional α-1,6-glycosidic linkages. Amylopectinconstitutes a complex mixture of glucose chains with different branchingpatterns.

Another important difference between amylose and amylopectin is theirmolecular weight. While amylose, depending on the origin of the starch,has a molecular weight of 5×10⁵-10⁶ Da, the molecular weight ofamylopectin is between 10⁷ and 10⁸ Da. The two macromolecules can bedistinguished on the basis of their molecular weight and their differentphysico-chemical properties, and the simplest way of visualizing this isthrough their different iodine-binding properties.

In addition to the amylose/amylopectin ratio and the phosphate content,the functional properties of starch are affected greatly by themolecular weight, the side-chain distribution pattern, the ioniccontent, the lipid and protein content, the mean granule size and thegranule morphology and the like. Important functional properties whichmay be mentioned in this context are solubility, the retrogradationbehaviour, the water-binding capacity, the film-forming properties,viscosity, the gelatinization properties, freeze-thaw-stability, acidstability, gel strength and the like. Granule size may also be ofimportance for various applications.

The skilled workers frequently resort to different methods to determinethe gelatinization properties, one of which is the final viscosity.Depending on the method used, absolute values in particular, but alsorelative values, may differ between one and the same starch sample. Arapid and effective method for analysing the gelatinization propertiesis the RVA analysis. Depending on the choice of the parameters and thetemperature profile in the RVA analysis, different RVA profiles areobtained for one and the same sample. It should be mentioned that insome cases different profiles were used in the prior art mentionedhereinbelow when determining the gelatinization properties.

An overview over different plant species with a reduction of the enzymesparticipating in starch biosynthesis can be found in Kossmann and Lloyd(2000, Critical Reviews in Plant Sciences 19(3), 171-126).

To date, plants have been described in which the activity of an SSIIIprotein (Abel et al., 1996, The Plant Journal 10(6), 981-991; Lloyd etal., 1999, Biochemical Journal 338, 515-521) or the activity of a BEIprotein (Kossman et al. 1991, Mol Gen Genet 230, 39-44); Safford et al.,1998, Carbohydrate Polymers 35, 155-168, or the activity of a BEIIprotein (Jobling et al., 1999, The Plant Journal 18(2): 163-171, or theactivity of a BEI and a BEII protein (Schwall et al., 2000, NatureBiotechnology 18, 551-554; WO 96/34968), or the activity of a BEI and anSSIII (WO 00/08184) protein are reduced.

In plants in which the activity of an SSIII protein is reduced, arelative shift of the amylopectin side chains from longer chains towardsshorter chains (Lloyd et al., 1999, Biochemical Journal 338, 515-521), a70% higher phosphate content, no changes in the amylose content (Abel etal., 1996, The Plant Journal 10(6), 9891-991) and a reduced finalviscosity in the RVA analysis (Abel, 1995, PhD Thesis at the FreieUniversität Berlin) are observed in comparison with correspondingwild-type plants. In such plants, which are also described in WO00/08184, a 197% increase in the phosphate content, 123% increase in theamylose content and a final viscosity in the RVA analysis which drops to76% of that of the wild type can be observed in the isolated starch incomparison with untransformed wild-type plants. Moreover, the gelstrength of the starch in question drops to 84% of the wild type.

The spectrophotometric analysis by the method of Morrison & Laignelet(1983, J. Cereal Sc. 1, 9-20) reveals an amylose content of up to amaximum of 89.14% (corresponding to 344% of the wild type) and a starchphosphate content which corresponds to up to a maximum of 522% of thephosphate content of starch isolated from corresponding wild-type plantsin plants with a reduced activity of both a BEI and a BEII protein. TheRVA analysis reveals a final viscosity value in these starches which isincreased up to a maximum of 237%. Moreover, the modified granulemorphology in starch grains isolated from such plants is distinguishedby the fact that the granules have large grooves in the centre of thegranule in question when viewed under the microscope under polarizedlight.

As a result, the skilled worker is familiar with plant cells and plantsand starches synthesized by them which have an increased amylose andphosphate content but whose final viscosity in the RVA analysis isincreased by not more than up to a maximum of 256% in comparison withwild-type plants which have not been genetically modified. Higher finalviscosities in the RVA analysis have not been achieved to date. However,this would be desirable since less starch solids have to be employed,for example when using such a starch as thickener, gelling agent orbinder, in order to achieve the desired effect. This allows for examplethe reduction of the amount of additives in human and animal foods, inhealthcare products and in cosmetics. It would also be possible toemploy smaller amounts of starch when using such a starch in glues,leading to reduced costs for example in making, for example, paper,cardboard and insulating board.

The present invention is thus based on the object of providing plantcells, plants and starch from suitable plant cells or plants with anincreased amylose content and an increased phosphate content and, in theRVA analysis, a final viscosity which is increased by at least 270%and/or an increased gel strength of the gelatinized starch and/or amodified granule morphology.

This object is achieved by providing the embodiments specified in thepatent claims.

A first aspect of the present invention thus relates to a plant cellwhich is genetically modified, the genetic modification leading to thereduction of the activity of one or more SSIII proteins occurringendogenously in the plant cell and to the reduction of the activity ofone or more BEI proteins which occur endogenously in the plant cell andto the reduction of the activity of one or more BEII proteins whichoccur endogenously in the plant cell in comparison to correspondingplant cells, of wild-type plants, which have not been geneticallymodified.

In this context, the genetic modification can be any geneticmodification which leads to a reduction of the activity of one or moreSSIII proteins which occur endogenously in the plant cell and to thereduction of the activity of one or more BEI proteins which occurendogenously in the plant cell and to the reduction of the activity ofone or more BEII proteins which occur endogenously in the plant cell incomparison to corresponding plant cells, of wild-type plants, which havenot been genetically modified.

For the purposes of the invention, the genetic modification mayencompass for example the generation of plant cells according to theinvention by subjecting one or more genes to mutagenesis. The type ofmutation is irrelevant as long as it leads to a reduction of theactivity of an SSIII protein and/or a BEI protein and/or a BEII protein.In connection with the present invention, the term “mutagenesis” isunderstood as meaning any type of mutation, such as, for example,deletions, point mutations (nucleotide substitutions), insertions,inversions, gene conversions or chromosome translocation.

In this context, the mutation can be generated by using chemical agentsor high-energy radiation (for example x-rays, neutron, gamma, UVradiation). Agents which can be employed for generating chemicallyinduced mutations, and the mutations generated thereby by the action ofthe mutagens in question, are described, for example, by Ehrenberg andHusain, 1981, (Mutation Research 86, 1-113), Müller, 1972 (BiologischesZentralblatt 91 (1), 31-48). The generation of rice mutants using gammarays, ethyl methanesulfonate (EMS), N-methyl-N-nitrosurea or sodiumazide (NaN3) is described, for example, in Jauhar and Siddiq (1999,Indian Journal of Genetics, 59 (1), 23-28), in Rao (1977), Cytologica42, 443-450), Gupta and Sharma (1990, Oryza 27, 217-219) and Satoh andOmura (1981, Japanese Journal of Breeding 31 (3), 316-326). Thegeneration of wheat mutants using NaN3 or maleic hydrazide is describedin Arora et al. (1992 Annals of Biology 8 (1), 65-69). An overview overthe generation of wheat mutants using different types of high-energyradiation and chemical agents is given in Scarascia-Mugnozza et al.(1993, Mutation Breeding Review 10, 1-28). Svec et al. (1998, CerealResearch Communications 26 (4), 291-396) describes the use ofN-ethyl-N-nitrosurea for generating mutants in triticale. The use of MMSand gamma radiation for generating millet mutations is described inShashidhara et al. (1990, Journal of Maharashtra AgricultureUniversities 15 (1), 20-23).

The generation of mutants in plant species whose propagation ispredominantly vegetatively was described for example for potatoes whichproduce a modified starch (Hovenkamp-Hermelink et al. (1987, Theoreticaland Applied Genetics 75, 217-221) and for mint with an increased oilyield/modified oil quality (Dwivedi et al., 2000, Journal of Medicinaland Aromatic Plant Sciences 22, 460-463). All of these methods aresuitable in principle for-generating the plant cells according to theinvention and the starch produced by them.

Mutations in the relevant genes, in particular in genes encoding a BEIprotein and/or a BEII protein and/or an SSIII protein, can be identifiedwith the aid of methods known to the skilled worker. Analyses based onhybridizations with probes (Southern blot), the amplification by meansof polymerase chain reaction (PCR), the sequencing of genomic sequencesin question, and the search for individual nucleotide substitutions, maybe employed in particular. One method of identifying mutations with theaid of hybridization patterns is, for example, the search forrestriction fragment length polymorphisms (RFLP) (Nam et al., 1989, ThePlant Cell 1, 699-705; Leister and Dean, 1993, The Plant Journal 4 (4),745-750). An example of a method based on PCR is the analysis ofamplified fragment length polymorphisms (AFLP) (Castiglioni et al.,1998, Genetics 149, 2039-2056; Meksem et al., 2001, Molecular Geneticsand Genomics 265, 207-214; Meyer et al., 1998, Molecular and GeneralGenetics 259,150-160). The use of amplified fragments cleaved with theaid of restriction endonucleases (cleaved amplified polymorphicsequences, CAPS) may also be used for identifying mutations (Koniecznyand Ausubel, 1993, The Plant Journal 4, 403-410; Jarvis et al., 1994,Plant Molecular Biology 24, 685-687; Bachem et al., 1996, The PlantJournal 9 (5), 745-753). Methods for determining SNPs have beendescribed, inter alia, by Qi et al. (2001, Nucleic Acids Research 29(22), e116) Drenkard et al. (2000, Plant Physiology 124, 1483-1492) andCho et al. (1999, Nature Genetics 23, 203-207). Methods which areparticularly suitable are those which permit a large number of plants tobe analysed within a short time for mutations in specific genes. Such amethod, known as TILLING (targetting induced local lesions in genomes),has been described by McCallum et al. (2000, Plant Physiology 123,439-442).

The use of all of these methods is suitable in principle for thepurposes of the present invention.

Hoogkamp et al. (2000, Potato Research 43, 179-189) have isolated stablepotato mutants which contain an amylose-free starch. These plants nolonger synthesize active enzyme for a granule-bound starch synthase(GBSS I). After subjecting these plants to another mutagenesis, thosewhich additionally have mutations in genes which are involved in starchbiosynthesis may be selected. Plants which synthesize starch withimproved characteristics might thus be generated. Using the suitablemethod, it is also possible to identify and isolate the plant cellsaccording to the invention which produce a starch according to theinvention.

Moreover, the plant cells according to the invention may also begenerated with the aid of homologous transposons, that is to saytransposons which are naturally present in the plant cells in question.A detailed description of this method is given hereinbelow.

All of the abovementioned methods are suitable in principle forgenerating plant cells according to the invention and the modifiedstarch synthesized by them. The present invention therefore also relatesto methods for generating genetically modified plant cells whichsynthesize a modified starch, this starch being characterized in that ithas an amylose content of at least 30%, in that it has an increasedphosphate content in comparison with starch from corresponding wild-typeplant cells which have not been genetically modified and in that it hasan increased final viscosity in the RVA analysis in comparison withstarch from corresponding wild-type plant cells which have not beengenetically modified.

A further aspect of the present invention relates to methods forgenerating a plant cell which synthesizes a modified starch, comprisingthe genetic modification of the plant cell, the genetic modificationleading to the reduction of the activity of one or more SSIII proteinswhich occur endogenously in the plant cell and to the reduction of theactivity of one or more BEI proteins which occur endogenously in theplant cell and to the reduction of the activity of one or more BEIIproteins which occur endogenously in the plant cell, in comparison withcorresponding plant cells, of wild-type plants, which have not beengenetically modified.

Yet a further aspect of the present invention relates to methods forgenerating a genetically modified plant which synthesizes a modifiedstarch, in which

-   a) a plant cell is generated as described above;-   b) a plant is regenerated from, or using, the plant cell generated    in accordance with a); and,-   c) if appropriate, further plants are generated from the plant    generated in accordance with step b).

In connection with the present invention, the term “geneticallymodified” means that the genetic information of the plant cell isaltered.

In this context, a reduction of the activity of one or more SSIIIproteins which occur endogenously in the plant cell and a reduction ofthe activity of one or more BEI proteins which occur endogenously in theplant cell and a reduction of the activity of one or more BEII proteinswhich occur endogenously in the plant cell is observed in the plantcells according to the invention in comparison with corresponding plantcells, of wild-type plants, which have not been genetically modified.

The genetic modifications for generating the plant cells according tothe invention can be performed simultaneously or in consecutive steps.In this context, each genetic modification can lead to the reduction ofthe activity of one or more SSIII proteins and/or one or more BEIproteins and/or one or more BEII proteins. The starting material may beeither wild-type plants or wild-type plant cells in which no previousgenetic modification in order to reduce the activity of one or moreSSIII proteins and/or one or more BEI proteins and/or one or more BEIIproteins has been performed, or else genetically modified plant cells orplants in which the activity of one or more SSIII proteins and/or one ormore BEI proteins and/or one or more BEII proteins has already beencarried out by genetic modification. If such genetically modified plants(plant cells) constitute the starting material, the geneticmodifications which are subsequently carried out preferably only relateto the activity of in each case one or more proteins whose activity hasnot been reduced yet (SSIII, BEI or BEII).

For example, a reduction of the expression of one or more SSIII geneswhich occur endogenously in the plant cell and a reduction of theexpression of one or more BEI genes which occur endogenously in theplant cell and a reduction of the expression of one or more BEII geneswhich occur endogenously in the plant cell and/or a reduction of theactivity of in each case one or more of the abovementioned proteinswhich occur in the plant cell is observed in genetically modified plantcells according to the invention in comparison with plant cells, ofwild-type plants, which have not been genetically modified.

For the purposes of the present invention, the term “reduction of theactivity” refers to a reduction of the expression of endogenous geneswhich encode SSIII, BEI and/or BEII proteins, and/or a reduction of theamount of SSIII, BEI and/or BEII protein in the cells and/or a reductionof the enzymatic activity of the SSIII, BEI and/or BEII proteins in thecells.

The reduction of the expression can be determined for example bymeasuring the amount of SSIII, BEI or BEII protein-encoding transcripts,for example by Northern blot analysis or RT-PCR. A reduction preferablymeans, in this context, a reduction of the amount of transcripts by atleast 50%, in particular by at least 70%, preferably by at least 85% andespecially preferably by at least 95% in comparison to correspondingcells which have not been genetically modified.

The reduction of the amount of SSIII, BEI and/or BEII proteins whichresults in a reduced activity of these proteins in the plant cells inquestion can be determined for example by immunological methods such asWestern blot analysis, ELISA (enzyme-linked immunosorbent assay) or RIA(radio-immune assay). In this context, a reduction preferably means areduction of the amount of SSIII, BEI and/or BEII protein by at least50%, in particular by at least 70%, preferably by at least 85% andespecially preferably by at least 95% in comparison to correspondingcells which have not been genetically modified.

In connection with the present invention, SSIII protein is understood asmeaning a class of soluble starch synthases(ADP-glucose-1,4-alpha-D-glucan-4-alpha-D-glucosyltransferase; EC2.4.1.21). Soluble starch synthases catalyze a glycosylation reaction,in which glucose residues of the substrate ADP-glucose are transferredto alpha-1,4-linked glucan chains, with formation of an alpha-1,4linkage (ADPglucose+{(1,4)-alpha-D-glucosyl}(N)<=>ADP+{(1,4)-alpha-D-glucosyl}(N+1)).

SSIII proteins are described, for example, by Marshall et al. (1996, ThePlant Cell 8, 1121-1135), Lie et al. (2000, Plant Physiology 123,613-624), Abel et al. (The Plant Journal 10(6); (1996); 981-991) and inWO 00/66745. The structure of SSIII proteins frequently shows a sequenceof domains. At the N terminus, SSIII proteins have a signal peptide forthe transport into plastids. Towards the C terminus, this is followed byan N-terminal region, an SSIII-specific region and a catalytic domain(Li et al., 2000, Plant Physiology 123, 613-624). Further analyses whichare based on primary sequence alignments, revealed that the potato SSIIIprotein has what is known as a carbohydrate binding domain (CBM). Thisdomain (Pfam motiv cbm 25) comprises the amino acids 377 to 437 of thesequence of the potato SSIII protein shown in Seq ID No. 2. Inconnection with the present invention, an SSIII protein is therefore tobe understood as meaning starch synthases which have at least 50%,preferably at least 60%, especially preferably at least 70%, morepreferably at least 80% and in particular at least 90% identity with thesequence shown in Seq ID No. 3.

The term homology, or identity, is understood as meaning the number ofagreeing amino acids (identity) with other proteins, expressed inpercent. The identity is preferably determined by comparing the Seq IDNo. 3 with other proteins with the aid of computer programmes. Ifsequences which are compared with each other are different in length,the identity is to be determined in such a way that the number of aminoacids which the short sequences shares with the longer sequencedetermines the percentage identity. The identity can be determinedroutinely by means of known computer programmes which are publiclyavailable such as, for example, ClustalW (Thompson et al., Nucleic AcidsResearch 22 (1994), 4673-4680). ClustalW is made publicly available byJulie Thompson and Toby Gibson, European Molecular Biology Laboratory,Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can likewise bedownloaded from various internet pages, inter alia the IGBMC (Insitut deGénétique et de Biologie Moléculaire et Cellulaire, B.P.163, 67404Illkirch Cedex, France and the EBI and all mirrored EBI internet pages(European Bioinformatics Institute, Wellcome Trust Genome Campus,Hinxton, Cambridge CB10 1SD, UK).

If the ClustalW computer programme Version 1.8 is used to determine theidentity between, for example, the reference protein of the presentapplication and other proteins, the following parameters are to be set:KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05,GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

One possibility of finding similar sequences is to carry out sequencedatabase researches. Here, one or more sequences are entered as what isknown as a query. This query sequence is then compared with sequencespresent in the selected databases using statistical computer programmes.Such database queries (blast searches) are known to the skilled workerand can be carried out at different suppliers. If, for example, such adatabase query is carried out at the NCBI (National Center forBiotechnology Information, the standard setting for the respectivecomparison query should be used. For protein sequences comparisons(blastp), these settings are: Limit entrez=not activated; Filler=lowcomplexity activated; Expect value=10; word size=3; Matrix=BLOSUM62; Gapcosts: Existence=11, Extension=1. The result of such a query is, amongother parameters, the degree of identity between the query sequence andthe similar sequences found in the databases.

Thus, an SSIII protein is to be understood as meaning, in connectionwith the present invention, starch synthases which, when using at leastone of the above-described methods for determining the identity with thesequence shown in Seq ID No. 3, have at least 50%, preferably at least60%, especially preferably at least 70%, more preferably at least 80%and in particular at least 90% identity.

For the purposes of the present invention, the term SSIII gene isunderstood as meaning a nucleic acid molecule (DNA, cDNA, RNA) whichencodes an SSIII protein, preferably from potato. Nucleic acid moleculesencoding an SSIII protein have been-described for a variety of plantspecies such as, for example, potato (Abel et al., The Plant Journal10(6); (1996); 981-991), wheat (WO 00/66745, Li et al., 2000, PlantPhysiology 123, 613-624; Genbank Acc. No AF258608; Genbank Acc. NoAF258609), maize (Gao et al., 1998, Plant Cell 10 (3), 399-412; GenbankAcc. No AF023159), Vignia (Genbank Acc. No AJ225088), rice (Genbank Acc.No AY100469; Genbank Acc. No AF43291) and Arabidopsis (Genbank Acc. NoAC007296).

For the purposes of the present invention, the term “branching enzyme”or “BE protein” (α-1,4-glucan: α-1,4-glucan-6-glycosyltransferase, E.C.2.4.1.18) is understood as meaning a protein which catalyzes atransglycosylation reaction in which α-1,4-linkages of an α-1,4-glucandonor are hydrolyzed and the α-1,4-glucan chains liberated in thisprocess are transferred to an α-1,4-glucan acceptor chain, where theyare converted into α-1,6 linkages.

The term “BEI protein” is to be understood as meaning, for the purposesof the present invention, an isoform I branching enzyme (branchingenzyme=BE). The BEI protein is preferably derived from potato plants.

In this context, isoform terminology relies on the nomenclature proposedby Smith-White and Preiss (Smith-White and Preiss, Plant Mol Biol. Rep.12, (1994), 67-71, Larsson et al., Plant Mol Biol. 37, (1998), 505-511).This nomenclature assumes that all enzymes which have a higher degree ofhomology (identity) at the amino acid level with the maize BEI protein(GenBank Acc. No. D11081; Baba et al., Biochem. Biophys. Res. Commun.181 (1), (1991), 87-94; Kim et al. Gene 216, (1998), 233-243) than tothe maize BEII protein (Genbank Acc. No AF072725, U65948) are referredto as isoform I branching enzymes, abbreviated to BEI proteins.

The term “BEII protein” is to be understood as meaning, for the purposesof the present invention, an isoform II branching enzyme (branchingenzyme=BE). This enzyme preferably originates from potato plants. Inconnection with the present invention, all enzymes which, at the aminoacid level, have a higher degree of homology (identity) with the maizeBEII protein (Genbank Acc. No AF072725, U65948) than with the maize BEIprotein (Genbank Acc. No. D 11081, AF 072724) shall be referred to asBEII protein.

The term “BEI gene” is understood as meaning, for the purposes of thepresent invention, a nucleic acid molecule (cDNA, DNA) which encodes a“BEI protein”, preferably a BEI protein from potato plants. Such nucleicacid molecules have been described for a large number of plants, forexample for maize (Genbank Acc. No. D 11081, AF 072724), rice (GenbankAcc. No. D11082), pea (Genbank Acc. No. X80010) and potato. Variousforms of the BEI gene, or the BEI protein, from potato have beendescribed, for example, by Khoshnoodi et al., Eur. J. Biochem. 242 (1),148-155 (1996), Genbank Acc. No. Y 08786 and by Kossmann et al., Mol.Gen. Genet. 230, (1991), 39-44). In potato plants, the BEI gene isexpressed predominantly in the tubers and to a very minor degree in theleaves (Larsson et al., Plant Mol. Biol. 37, (1998), 505-511).

The term “BEII gene” is to be understood as meaning, for the purposes ofthe present invention, a nucleic acid molecule (for example cDNA, DNA)which encodes a “BEII protein”, preferably a BEII protein from potatoplants. Such nucleic acid molecules have been described for a largenumber of plants, for example for potato (GenBank Acc. No. AJ000004,AJ011888, AJ011889, AJ011885, AJ011890, EMBL GenBank A58164), maize (AF072725, U65948), barley (AF064561), rice (D16201) and wheat (AF 286319).In potato plants, the BEII gene is expressed predominantly in the tubersand to a very minor degree in the leaves (Larsson et al., Plant Mol.Biol. 37, (1998), 505-511).

The term “transgenic” is to be understood as meaning, in the presentcontext, that the genetic information of the plant cells according tothe invention deviates from corresponding plant cells which have notbeen genetically modified owing to the introduction of a foreign nucleicacid molecule or several foreign nucleic acid molecules into the cell.

In a further embodiment of the present invention, the geneticmodification of the transgenic plant cell according to the inventionconsists in the introduction of one or more foreign nucleic acidmolecules whose presence and/or expression leads to the reduction of theactivity of SSIII and BEI and BEII proteins in comparison tocorresponding plant cells, of wild-type plants, which have not beengenetically modified. Specifically, the term “genetic manipulation” isunderstood as meaning the introduction of homologous and/or heterologousnucleic acid molecules and/or foreign nucleic acid molecules which havebeen subjected to mutagenesis into a plant cell, where said introductionof these molecules leads to the reduction of the activity of an SSIIIprotein and/or a BEI protein and/or BEII protein.

The term “foreign nucleic acid molecule” or “of foreign nucleic acidmolecules” is understood as meaning, for the purposes of the presentinvention, such a molecule which either does not occur naturally in theplant cells in question, or which does not naturally occur in the plantcells in the specific spatial arrangement, or which is localized at asite in the genome of the plant cell where it does not occur naturally.The foreign nucleic acid molecule is preferably a recombinant moleculewhich consists of various elements whose combination, or specificspatial arrangement, does not naturally occur in plant cells.

The foreign nucleic acid molecule(s) which is, or are, used for thegenetic modification may take the form of a hybrid nucleic acidconstruct or of several separate nucleic acid constructs, in particularof what are known as single, dual and triple constructs. Thus, theforeign nucleic acid molecule may be, for example, what is known as a“triple construct”, which is understood as meaning one single vector forplant transformation which contains not only the genetic information forinhibiting the expression of one or more endogenous SSIII genes, butalso the genetic information for inhibiting the expression of one ormore BEI genes and of one or more BEII genes, or whose presence, orexpression, leads to the reduction of the activity of one or more SSIII,BEI and BEII proteins.

In a further embodiment, the foreign nucleic acid molecule may be whatis known as a “dual construct”, which is understood as meaning a vectorfor plant transformation which contains the genetic information forinhibiting the expression of two out of the three target genes (SSIII,BEI, BEII gene) or whose presence, or expression, leads to the reductionof the activity of two out of the three target proteins (SSIII, BEI,BEII proteins). The inhibition of the expression of the third targetgene and/or the reduction of the activity of the third target protein iseffected, in this embodiment of the invention, with the aid of aseparate, foreign nucleic acid molecule which contains the relevantgenetic information for inhibiting this third target gene.

In a further embodiment of the invention, it is not a triple constructwhich is introduced into the genome of the plant cell, but severaldifferent foreign nucleic acid molecules are introduced, one of theseforeign nucleic acid molecules being, for example, a DNA molecule whichconstitutes, for example, a cosuppression construct which brings about areduction of the expression of one or more endogenous SSIII genes, and afurther foreign nucleic acid molecule being a DNA molecule whichencodes, for example, an antisense RNA which brings about a reduction ofthe expression of one or more endogenous BEI and/or BEII genes. Whenconstructing the foreign nucleic acid molecules, however, the use of anycombination of antisense, cosuppression, ribozyme and double-strandedRNA constructs or in-vivo mutagenesis which leads to a simultaneousreduction of the gene expression of endogenous genes encoding one ormore SSIII, BEI and BEII proteins, or which leads to a simultaneousreduction of the activity of one or more SSIII, BEI and BEII proteins,is also suitable in principle.

The foreign nucleic acid molecules can be introduced into the genome ofthe plant cell either simultaneously (“cotransformation”) or else oneafter the other, i.e. in succession at different times(“supertransformation”).

The foreign nucleic acid molecules can also be introduced into differentindividual plants of one species. This may give rise to plants in whichthe activity of one target protein, or two target proteins, (BEI, BEII,SSIII) is reduced. Subsequent hybridizing may then give rise to plantsin which the activity of all three of the target proteins is reduced.

Instead of a wild-type plant cell or plant, a mutant which isdistinguished by already showing a reduced activity of one or moretarget proteins (BEI, BEII, SSIII) may further be used for introducing aforeign nucleic acid molecule or for generating the plant cells orplants according to the invention. The mutants may take the form ofspontaneously occurring mutants or else of mutants which have beengenerated by the specific application of mutagens. Possibilities ofgenerating such mutants have been described further above.

The plant cells according to the invention and their starch can begenerated, or produced, by using what is known as insertion mutagenesis(review article: Thorneycroft et al., 2001, Journal of experimentalBotany 52 (361), 1593-1601). Insertion mutagenesis is to be understoodas meaning, in particular, the insertion of transposons or what is kownas transfer DNA (T-DNA) into a gene encoding a BEI protein and/or BEIIprotein and/or an SSIII protein, thus reducing the activity of saidproteins in the cell in question.

The transposons may take the form of transposons which occur naturallyin the cell (endogenous transposons) or else those which do not occurnaturally in said cell but have been introduced into the cell by meansof recombinant methods, such as, for example, by transforming the cell(heterologous transposons). Modifying the expression of genes by meansof transposons is known to the skilled worker. A review of theutilization of endogenous and heterologous transposons as tools in plantbiotechnology can be found in Ramachandran and Sundaresan (2001, PlantPhysiology and Biochemistry 39, 234-252). The possibility of identifyingmutants in which specific genes have been inactivated by transposoninsertion mutagenesis can be found in a review by Maes et al. (1999,Trends in Plant Science 4 (3), 90-96). The generation of rice mutantswith the aid of endogenous transposons is described by Hirochika (2001,Current Opinion in Plant Biology 4, 118-122). The identification ofmaize genes with the aid of endogenous retrotransposons is shown, forexample, in Hanley et al. (2000, The Plant Journal 22 (4), 557-566). Thepossibility of generating mutants with the aid of retrotransposons andmethods for identifying mutants are described by Kumar and Hirochika(2001, Trends in Plant Science 6 (3), 127-134). The activity ofheterologous transposons in different species has been described bothfor dicotyledonous and for monocotyledonous plants, for example for rice(Greco et al., 2001, Plant Physiology 125, 1175-1177; Liu et al., 1999,Molecular and General Genetics 262, 413-420; Hiroyuki et al., 1999, ThePlant Journal 19 (5), 605-613; Jeon and Gynheung, 2001, Plant Science161, 211-219), barley (2000, Koprek et al., The Plant Journal 24 (2),253-263), Arabidopsis thaliana (Aarts et al., 1993, Nature 363, 715-717,Schmidt and Willmitzer, 1989, Molecular and General Genetics 220, 17-24;Altmann et al., 1992, Theoretical and Applied Gentics 84, 371-383;Tissier-et al., 1999, The Plant Cell 11, 1841-1852), tomato (Beizile andYoder, 1992, The Plant Journal 2 (2), 173-179) and potato (Frey et al.,1989, Molecular and

General Genetics 217, 172-177; Knapp et al., 1988, Molecular and GeneralGenetics 213, 285-290).

In principle, the plant cells and plants according to the invention, andthe starch produced by them, can be generated, or produced, with the aidof both homologous and heterologous transposons, the use of homologoustransposons also including those transposons which are already naturallypresent in the plant genome.

T-DNA insertion mutagenesis is based on the fact that certain segments(T-DNA) of Ti plasmids from Agrobacterium are capable of integratinginto the genome of plant cells. The site of integration into the plantchromosome is not fixed but may take place at any position. If the T-DNAintegrates in a segment of the chromosome which constitutes a genefunction, this may lead to a modification of the gene expression andthus also to an altered activity of a protein encoded by the gene inquestion. In particular, the integration of a T-DNA into the codingregion of a protein frequently means that the protein in question can nolonger be synthesized in active form, or not at all, by the cell inquestion. The use of T-DNA insertions for the generation of mutants isdescribed, for example, for Arabidopsis thaliana (Krysan et al., 1999,The Plant Cell 11, 2283-2290; Atipiroz-Leehan and Feldmann, 1997, Trendsin genetics 13 (4), 152-156; Parinov and Sundaresan, 2000, CurrentOpinion in Biotechnology 11, 157-161) and rice (Jeon and An, 2001, PlantScience 161, 211-219; Jeon et al., 2000, The Plant Journal 22 (6),561-570). Methods for identifying mutants which have been generated withthe aid of T-DNA insertion mutagenesis are described, inter alia, byYoung et al., (2001, Plant Physiology 125, 513-518), Parinov et al.(1999, The Plant cell 11, 2263-2270), Thorneycroft et al. (2001, Journalof Experimental Botany 52, 1593-1601), and McKinney et al. (1995, ThePlant Journal 8 (4),613-622).

In principle, T-DNA mutagenesis is suitable for generating the plantcells according to the invention and for producing the starch producedby them.

In a further embodiment of the present invention, the presence and/orthe expression of one or more foreign nucleic acid molecules leads tothe inhibition of the expression of endogenous genes which encode SSIIIproteins, BEI proteins and BEII proteins.

The plant cells according to the invention can be generated by variousmethods with which the skilled worker is familiar, for example by thosewhich lead to an inhibition of the expression of endogenous genesencoding an SSIII, BEI or BEII protein. They include, for example, theexpression of a corresponding antisense RNA or a double-stranded RNAconstruct, the provision of molecules or vectors which confer acosuppression effect, the expression of a suitably constructed ribozymewhich specifically cleaves transcripts encoding an SSIII, BEI or BEIIprotein, or what is known as “in-vivo mutagenesis”. Moreover, thereduction of the SSIII and/or BEI and/or BEII activity in the plantcells may also be brought about by the simultaneous expression of senseand antisense RNA molecules of the specific target gene to be repressed,preferably the SSIII and/or BEI and/or BEII gene. The skilled worker isfamiliar with these methods.

Moreover, it is known that the generation in planta of double-strandedRNA molecules of promoter sequences in trans can lead to methylation andtranscriptional inactivation of homologous copies of this promoter(Mette et al., EMBO J. 19, (2000), 5194-5201).

Other methods for reducing the activity of proteins are describedhereinbelow.

All of these methods are based on the introduction of one or moreforeign nucleic acid molecules into the genome of plant cells.

To inhibit gene expression by means of antisense or cosuppressiontechnology it is possible to use, for example, a DNA molecule whichencompasses all of the sequence encoding an SSIII and/or BEI and/or BEIIprotein including any flanking sequences which may be present, or elseDNA molecules which only encompass parts of the coding sequence, whichmust be long enough in order to bring about an antisense effect, orcosuppression effect, in the cells. Sequences which are suitablegenerally have a minimum length of not less than 15 bp, preferably alength of 100-500 bp, and for effective antisense or cosuppressioninhibition in particular sequences which have a length of over 500 bp.

Another possibility which is suitable for antisense or cosuppressionapproaches is the use of DNA sequences with a high degree of homologywith the endogenous sequences which encode SSIII, BEI or BEII proteinsand which occur endogenously in the plant cell. The minimum degree ofhomology should exceed approximately 65%. The use of sequences withhomology levels of at least 90%, in particular between 95 and 100%, isto be preferred.

The use of introns, i.e. noncoding regions of genes, which encode SSIII,BEI and/or BEII proteins is also feasible for achieving an antisense orcosuppression effect.

The use of intron sequences for inhibiting the gene expression of geneswhich encode starch biosynthesis proteins has been described in theinternational patent applications WO97/04112, WO97/04113, WO98/37213,WO98/37214.

The skilled worker is familiar with methods for achieving an antisenseand cosuppression effect. The cosuppression inhibition method has beendescribed, for example, in Jorgensen (Trends Biotechnol. 8 (1990),340-344), Niebel et al., (Curr. Top. Microbiol. Immunol. 197 (1995),91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995),43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159),Vaucheret et al., (Mol. Gen. Genet. 248 (1995), 311-317), de Borne etal. (Mol. Gen. Genet. 243 (1994), 613-621).

The expression of ribozymes for reducing the activity of specificenzymes in cells is also known to the skilled worker and described, forexample, in EP-B1 0321201. The expression of ribozymes in plant cellshas been described, for example, in Feyter et al. (Mol. Gen. Genet. 250,(1996), 329-338).

Moreover, the reduction of the SSIII and/or BEI and/or BEII activity inthe plant cells may also be achieved by what is known as “in-vivomutagenesis”, where an RNA-DNA oligonucleotide hybrid (“chimeroplast”)is introduced into cells by means of transforming cells (Kipp, P. B. etal., Poster Session at the “5^(th) International Congress of PlantMolecular Biology, 21-27 Sep. 1997, Singapore; R. A. Dixon and C. J.Arntzen, Meeting report on “Metabolic Engineering in Transgenic Plants”,Keystone Symposia, Copper Mountain, Colo., USA, TIBTECH 15, (1997),441-447; International Patent Application WO 9515972; Kren et al.,Hepatology 25, (1997), 1462-1468; Cole-Strauss et al., Science 273,(1996), 1386-1389; Beetham et al., 1999, PNAS 96, 8774-8778).

Part of the DNA component of the RNA-DNA oligonucleotide is homologouswith a nucleic acid sequence of an endogenous SSIII, BEI and/or BEIIgene, but contains a mutation in comparison with the nucleic acidsequence of an endogenous SSIII, BEI and/or BEII gene or contains aheterologous region which is surrounded by the homologous regions.

Owing to base pairing of the homologous regions of the RNA-DNAoligonucleotide and of the endogenous nucleic acid molecule, followed byhomologous recombination, the mutation or heterologous region containedin the DNA component of the RNA-DNA oligonucleotide can be transferredinto the genome of a plant cell. This leads to a reduction of theactivity of one or more SSIII, BEI and/or BEII proteins.

Moreover, the reduction of the SSIII and/or BEI and/or BEII activity inthe plant cells may also be caused by the simultaneous expression ofsense and antisense RNA molecules of the specific target gene to berepressed, preferably the SSIII and/or BEI and/or BEII gene.

This may be achieved for example by the use of chimeric constructs whichcontain “inverted repeats” of the respective target gene or parts of thetarget gene. The chimeric constructs encode sense and antisense RNAmolecules of the target gene in question. Sense and antisense RNA aresynthesized simultaneously in planta as one RNA molecule, it beingpossible for sense and antisense RNA to be separated from each other bya spacer and to form a double-stranded RNA molecule.

It has been demonstrated that the introduction of inverted-repeat DNAconstructs in the genome of plants is a highly effective method forrepressing the genes corresponding to the inverted-repeat DNA constructs(Waterhouse et al., Proc. Natl. Acad. Sci. USA 95, (1998), 13959-13964;Wang and Waterhouse, Plant Mol. Biol. 43, (2000), 67-82;Singh et al.,Biochemical Society Transactions Vol. 28 part 6 (2000), 925-927; Liu etal., Biochemical Society Transactions Vol. 28 part 6 (2000), 927-929);Smith et al., (Nature 407, (2000), 319-320; International PatentApplication WO99/53050 A1). Sense and antisense sequences of the targetgene(s) may also be expressed separately from one another by means ofidentical or different promoters (Nap, J-P et al, 6^(th) InternationalCongress of Plant Molecular Biology, Quebec, 18-24 Jun., 2000; PosterS7-27, Session S7).

The reduction of the SSIII and/or the BEI and/or the BEII activity inthe plant cells can thus also be achieved by generating double-strandedRNA molecules of SSIII and/or BEI and/or BEII genes. To this end, it ispreferred to introduce, into the genome of plants, inverted repeats ofDNA molecules of SSIII and/or BEI and/or BEII genes or cDNAs, the DNAmolecules to be transcribed (SSIII, BEI or BEII gene or cDNA, orfragments of these genes or cDNAs) being under the control of a promoterwhich governs the expression of said DNA molecules.

Moreover, it is known that the formation of double-stranded RNAmolecules of promoter DNA molecules in plants in trans can lead tomethylation and transcriptional inactivation of homologous copies ofthese promoters, hereinbelow referred to as target promoters (Mette etal., EMBO J. 19, (2000), 5194-5201).

Thus, it is possible, via the inactivation of the target promoter, toreduce the gene expression of a specific target gene (for example SSIII,BEI or BEII gene) which is naturally under the control of this targetpromoter.

This means that the DNA molecules which encompass the target promotersof the genes to be repressed (target genes) are in this case—in contrastto the original function of promoters in plants—not used as controlelements for the expression of genes or cDNAs, but as transcribable DNAmolecules themselves.

To generate the double-stranded target promoter RNA molecules in planta,where they may be present in the form of RNA hairpin molecules, it ispreferred to use constructs which contain inverted repeats of the targetpromoter DNA molecules, the target promoter DNA molecules being underthe control of a promoter which governs the gene expression of saidtarget promoter DNA molecules. These constructs are subsequentlyintroduced into the genome of plants. Expression of the inverted repeatsof said target promoter DNA molecules leads to the formation ofdouble-stranded target promoter RNA molecules in planta (Mette et al.,EMBO J. 19, (2000), 5194-5201). The target promoter can thus beinactivated.

The reduction of the SSIII and/or BEI and/or BEII activity in the plantcells can thus also be achieved by generating double-stranded RNAmolecules of promoter sequences of SSIII and/or BEI and/or BEII genes.To this end, it is preferred to introduce, into the genome of plants,inverted repeats of promoter DNA molecules of SSIII and/or BEI and/orBEII promoters, the target promoter DNA molecules to be transcribed(SSIII, BEI and/or BEII promoter) being under the control of a promoterwhich governs the expression of said target promoter DNA molecules.

The skilled worker furthermore knows to achieve the activity of one ormore SSIII, BEI and/or BEII proteins by expressing nonfunctionalderivatives, in particular trans-dominant mutants, of such proteinsand/or by expressing antagonists/inhibitors of such proteins.

Antagonists/inhibitors of such proteins encompass for exampleantibodies, antibody fragments or molecules with similar bindingcharacteristics. For example, a cytoplasmic scFv antibody was employedfor modulating the activity of the phytochrome A protein in geneticallymodified tobacco plants (Owen, Bio/Technology 10 (1992), 790-4; Review:Franken, E, Teuschel, U. and Hain, R., Current Opinion in Biotechnology8, (1997), 411-416; Whitelam, Trends Plant Sci. 1 (1996), 268-272).

Useful promoters for the expression of nucleic acids which reduce theactivity of a target gene are, for example, the promoter of thecauliflower mosaic virus 35S RNA and the maize ubiquitin promoter forconstitutive expression, the patatin gene promoter B33 (Rocha-Sosa etal., EMBO J. 8 (1989), 23-29), the MCPI promoter of the potatometallocarbopeptidase inhibitor gene (Hungarian Patent ApplicationHU9801674) or the potato GBSSI promoter (International PatentApplication WO 92/11376) for tuber-specific expression in potatoes, or apromoter which ensures expression uniquely in photosynthetically activetissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl.Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),2445-2451), the Ca/b promoter (see, for example, U.S. Pat. No.5,656,496, U.S. Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad.Sci. USA 89, (1992), 3654-3658) and the Rubisco SSU promoter (see, forexample, U.S. Pat. No. 5,034,322, U.S. Pat. No. 4,962,028), or, forendosperm-specific expression, the glutelin promoter (Leisy et al.,Plant Mol. Biol. 14, (1990), 41-50; Zheng et al., Plant J. 4, (1993),357-366; Yoshihara et al., FEBS Lett. 383, (1996), 213-218), theShrunken-1 promoter (Werr et al., EMBO J. 4, (1985), 1373-1380), thewheat HMG promoter, the USP promoter, the phaseolin promoter orpromoters of zein genes from maize (Pedersen et al., Cell 29, (1982),1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93).

The expression of the foreign nucleic acid molecule(s) is particularlyadvantageous in those plant organs which store starch. Examples of suchorgans are the tuber of the potato plant or the kernels, or endosperm,of maize, wheat or rice plants. This is why it is preferred to usepromoters which confer expression in these organs.

However, it is also possible to use promoters which are activated onlyat a point in time determined by external factors (see, for example, WO93/07279). Promoters which may be of particular interest in this contextare promoters of heat-shock proteins, which permit simple induction.Others which can be [lacuna] are seed-specific promoters such as, forexample, the Vicia faba USP promoter, which ensures seed-specificexpression in Vicia faba and other plants (Fiedler et al., Plant Mol.Biol. 22, (1993), 669-679; Bäumlein et al., Mol. Gen. Genet. 225,(1991), 459-467). Fruit-specific promoters such as, for example, thosedescribed in WO91/01373 may furthermore also be employed.

Another element which may be present is a termination sequence, whichserves for the correct termination of transcription and for the additionof a poly-A tail to the transcript, which is believed to have a functionin stabilizing the transcripts. Such elements are described in theliterature (cf., for example, Gielen et al., EMBO J. 8 (1989), 23-29)and can be substituted as desired.

The transgenic plant cells according to the invention synthesize amodified starch whose physico-chemical properties, in particular theamylose content and the amylose/amylopectin ratio, the phosphoruscontent, the viscosity behaviour, the gel strength, the granule sizeand/or the granule morphology is modified in comparison with starchsynthesized in wild-type plants so that it is better suited to specificuses.

The present invention therefore also relates to a genetically modifiedplant cell according to the invention, in particular to a transgenicplant cell which synthesizes a modified starch.

Surprisingly, it has been found that the starch composition in the plantcells according to the invention is modified in such a way that theamylose content amounts to at least 30% and the phosphate content isincreased and the end viscosity in the RVA analysis is increased incomparison with starch from plant cells from corresponding wild-typeplants, so that this starch is better suited to specific uses.

In particular the starches according to the invention have the advantagethat they gelatinize completely under standard conditions despite theincreased amylose content. This markedly improves the processability ofthe starch in comparison with other starches with an increased amylosecontent. An increased temperature or increased pressure is therefore notnecessary for gelatinizing the starch according to the invention. Thisis why the use of specific apparatuses such as, for example, jetcookers, extruders or autoclaves can be dispensed with when breakingdown these starches. Another advantage of the starches according to theinvention is that, when subjected to processing with hot rollers, theymay be applied to the latter in the form of a suspension. Other starcheswith an increased amylose content would gelatinize, when subjected tothis type of processing, to a limited extent only, if at all, and wouldnot be capable of being applied to the rollers in question in the formof a paste or film.

The starches according to the invention are particularly suitable forall applications where the thickening ability, the gellingcharacteristics or the binding characteristics of added substances areof importance. The starch according to the invention is thereforeparticularly suitable for the production of foodstuffs such as, forexample, baked goods, instant food, blancmange, soups, confectionery,chocolate, icecream, batter for fish or meat, frozen desserts orextruded snacks. Moreover, the starch according to the invention issuitable for the production of glues, for applications in textileprocessing, as additive to building materials, for applications in thefield of animal nutrition, as additive for cosmetics, and inpapermaking.

The starch which has been isolated from plant cells according to theinvention is particularly suitable for the production of pregelatinizedstarch. Pregelatinized starches are physically modified starches whichare produced predominantly by wet-heat treatment. As opposed to nativestarch, they form dispersions/pastes or gels with cold water, dependingon the concentration of-the pregelatinized starch used and as a functionof the starch type used for producing the pregelatinized starch. Owingto these characteristics, a series of possible applications exist forpregelatinized starches in the food industry and in addition in manyfields of industry. The use of pregelatinized starch, also referred toas cold-swelling starch, instead of native starch frequently has theadvantage that production processes can be simplified and shortened.

The production of, for example, instant desserts and instant blancmangerequires pregelatinized starches which, after being stirred into coldfluid such as, for example, water or milk, form firm gels in a shorttime as is the case with, for example, a blancmange which requiresboiling. These demands are not met by the commercial pregelatinizedstarches made with wheat starch, potato starch or corn starch. To obtainthe abovementioned characteristics, additives to the pregelatinizedstarch such as gelatin, alginate, carrageenan and/or inorganic salts arerequired in the case of the pregelatinized starches which are currentlycommercially available. This addition of what are known as adjuvants isnot required for example after the production of pregelatinized starchesusing starches according to the invention which are isolated from plantcells according to the invention.

The present invention also relates to a plant cell according to theinvention with a modified starch with modified granule morphology.

For the purposes of the present invention, the term granule morphologyis intended to refer to size and surface structure of native starchgranules. Starch is stored in the storage organs such as, for example,tubers, roots, embryos or endosperm of plants, as a crystallinestructure in granular form. Starch granules in which these granularstructures are retained after the starch has been isolated from plantcells are referred to as native starch. The mean granule size(determined by the method described hereinbelow) of the native starchaccording o the invention is markedly lower than that of native starchisolated from wild-type plants. In the scanning electron micrograph (seeFIGS. 4 and 5) it can be seen clearly that, surprisingly, native starchgranules according to the invention have a rough surface with manypores. The surface structure of native starch granules isolated fromwild-type plants, in contrast, is predominantly smooth in structure andno pores are discernible.

Both the presence of smaller granules and the rough surface with itspores lead to the fact that the surface area of starch granulesaccording to the invention is considerably larger—at the samevolume—than the surface area of starch granules isolated from wild-typeplants. The starch according to the invention is therefore particularlysuited to the use as carrier for, for example, flavourings,pharmacologically active substances, prebiotics, probioticmicroorganisms, enzymes or colorants. These starches are alsoparticularly suitable for coagulating substances and in papermaking.

A further possible application for the starches according to theinvention is in the field of drilling for raw materials. Thus, whendrilling for crude oil, adjuvants and/or lubricants must be employedwhich avoid overheating of the drill or drill column. Owing to itsparticular gelatinization properties, the starch according to theinvention is therefore also particularly suited to the use in thisfield.

The present invention also relates to a plant cell according to theinvention which contains a modified starch with an amylose content of atleast 30% and which has an increased phosphate content and an increasedfinal viscosity in the RVA analysis in comparison with starch fromcorresponding plant cells, from wild-type plants, which have not beengenetically modified.

In connection with the present inveniton, the amylose content isdetermined by the method of Hovenkamp-Hermelink et al. (Potato Research31, (1988), 241-246) described further below for potato starch. Thismethod may also be applied to isolated starches from other plantspecies. Methods for isolating starches are known to the skilled worker.

For the purposes of the present invention, “phosphate content” of thestarch refers to the content of phosphate covalently bonded in the formof starch phosphate monoesters.

In connection with the present invention, the term “increased phosphatecontent” means that the total phosphate content of covalently bondedphosphate and/or the phosphate content in C-6 position of the starchsynthesized in the plant cells according to the invention is increased,by preference by at least 270%, more preferably by at least 300%,especially preferably by at least 350% in comparison with starch fromplant cells of corresponding wild-type plants.

For the purposes of the present invention, the term “phosphate contentin C6 position” is understood as meaning the content of phosphate groupswhich are bonded at the carbon atom position “6” of the glucose monomersof the starch. In principle, the positions C2, C3 and C6 of the glucoseunits can be phosphorylated in starch in vivo. In connection with thepresent invention, the determination of the phosphate content in C6position (=C6-P content) can be carried out via the determination ofglucose-6-phosphate by means of a visual-enzymatic test (Nielsen et al.,Plant Physiol. 105, (1994), 111-117) (see below).

In connection with the present invention, the term “total phosphatecontent” of the starch refers to the content of phosphate boundcovalently in C2, C3 and C6 position of the glucose units in the form ofstarch phosphate monoesters. The content of phosphorylated non-glucanssuch as, for example, phospholipids, does not come under the term “totalphosphate content” in accordance with the invention. Phosphorylatednon-glucans must therefore be removed quantitatively before determiningthe total phosphate content. Methods for separating the phosphorylatednon-glucans (for example phospholipids) and the starch are known to theskilled worker. Methods for determining the total phosphate content areknown to the skilled worker and described hereinbelow.

In a further embodiment of the invention, the plant cells according tothe invention synthesize a starch which have a phosphate content of40-120 nmol, in particular 60-110 nmol, preferably 80-100 C6-P per mgstarch in C6 position of the glucose monomers of the starch.

A protocol for carrying out the RVA analysis is described further below.Mention must be made in particular that the RVA analysis of potatostarches frequently operates with an 8% starch suspension (w/w). Thedocumentation included with the apparatus “RVAsuper3” (instructions,Newport Scientific Pty Ltd., Investment Support Group, Warded NSW 2102,Australia) recommends a suspension containing approximately 10% ofstarch for the analysis of potato starch.

Surprisingly, it has been found in the case of the starch from potatoplants in relation to the present invention, that it was not possible touse an 8% starch suspension (2 g of starch in 25 ml of water) for theanalysis since the final viscosity achieved values beyond the range ofthe apparatus. This is why only 6% starch suspensions (1.5 g of starchin 25 ml of water) were employed for the RVA analysis instead of 8%starch suspensions. In connection with the present invention, “increasedend viscosity in the RVA analysis” is therefore understood as meaning anincrease by at least 150%, especially by at least 200%, in particular byat least 250%, in comparison with wild-type plants which have not beengenetically modified. The increase of the end viscosities relates to 6%starch suspensions in this context.

In connection with the present invention, a potato starch is furthermoreunderstood as meaning one with an at least 300 RVU, especially 400 RVU,in particular 500 RVU final viscosity in the RVA analysis with a 6%starch content. The determination of the RVU values will be discussed indetail hereinbelow.

In a further preferred embodiment, the present invention relates toplant cells according to the invention which synthesize a modifiedstarch which, after gelatinization in water, forms a gel with anincreased gel strength in comparison with a gel made with starch ofcorresponding wild-type plant cells which have not been geneticallymodified.

For the purposes of the present invention, the term “increased gelstrength” is understood as meaning an increase of the gel strength bypreference by at least 300%, in particular by at least 500%, morepreferably by at least 700% and especially preferably by at least 800%,up to a maximum of not more than 2000% or by not more than 1500% incomparison with the gel strength of starch from corresponding wild-typeplant cells which have not been genetically modified.

In connection with the present invention, the gel strength shall bedetermined with the aid of a Texture Analyser under the conditionsdescribed hereinbelow.

To prepare starch gels, the crystalline structure of native starch mustfirst be destroyed by heating in aqueous suspension with constantstirring. This was carried out with the aid of a Rapid Visco Analyser(Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW2102, Australia). As already mentioned further above, the 8% starchsuspension was replaced by an only 6% starch suspension in the case ofstarch from potato plants since the final viscosities of the 8%suspensions were outside the operating range of the apparatus. Todetermine gel strength, the starch suspensions gelatinized in the RapidVisco Analyser were stored over a certain period and then subjected toanalysis using a Texture Analyser. Accordingly, 8% gelatinized starchsuspensions were also replaced by 6% gelatinized starch suspensions fordetermining gel strength.

In a further embodiment of the present invention, the modified starchsynthesized in the plant cells according to the invention isdistinguished not only by an increased amylose content in comparisonwith starch from corresponding wild-type plants and an increasedphosphate content and an increased final viscosity in the RVA analysis,but also by a modified side chain distribution.

In a further embodiment, the present invention thus relates to plantcells according to the invention which synthesize a modified starch, themodified starch being characterized by a modified side chaindistribution.

In one embodiment of the present invention, the term “modified sidechain distribution” is understood as meaning a reduction of the amountof short side chains with a DP (=degree of polymerization) of 6 to 11 byat least 10%, preferably by at least 15%, in particular by at least 30%and especially preferably by at least 50% in comparison with the amountof short side chains with a DP of 6 to 11 of amylopectin from wild-typeplants and/or an increase in the content of short side chains with a DPof 6 to 22 by at least 5%, preferably by at least 10%, in particular byat least 15% and especially preferably by at least 30% in comparisonwith the amount of short side chains with a DP of 16 to 22 ofamylopectin from wild-type plants.

The amount of short side chains is determined via determining thepercentage of a specific side chain in the total of all side chains. Thetotal of all side chains is determined via determining the total areaunder the peaks which represent the degrees of polymerization of DP 6 to26 in the HPLC chromatogram. The percentage of a particular side chainin the total of all side chains is determined via determining the ratioof the area under the peak which represents this side chain in the HPLCchromatogram to the total area. A programme which may be used fordetermining the peak areas is, for example, Chromelion 6.20 from Dionex,USA.

In a further embodiment of the present invention, the modified starchsynthesized in the plant cells according to the invention isdistinguished not only by an increased amylose content in comparisonwith starch from corresponding wild-type plants and an increasedphosphate content and an increased final viscosity in the RVA analysis,but also by a modified “side chain profile DP 12 to 18” and/or by amodified “side chain profile DP 19 to 24” and/or by a modified “sidechain profile DP 25 to 30” and/or by a modified “side chain profile DP37 to 42” and/or by a modified “side chain profile DP 62 to 123”.

In connection with the present invention, the term modified “side chainprofile DP 12 to 18” is understood as meaning a reduction of the amountof amylopectin side chains with a DP of 12 to 18 by at least 25%,preferably by at least 35%, especially preferably by at least 45% andvery especially preferably by at least 55% in comparison with the amountof amylopectin side chains with a DP of 12 to 18 from wild-type plants.

In connection with the present invention, the term modified “side chainprofile DP 19 to 24” is understood as meaning a reduction of the amountof amylopectin side chains with a DP of 19 to 24 by at least 10%,preferably by at least 20% and especially preferably by at least 30% incomparison with the amount of amylopectin side chains with a DP of 19 to24 from wild-type plants.

In connection with the present invention, the term modified “side chainprofile DP 25 to 30” is understood as meaning a reduction of the amountof amylopectin side chains with a DP of 25 to 30 by at least 5% incomparison with the amount of amylopectin side chains with a DP of 25 to30 from wild-type plants.

In connection with the present invention, the term modified “side chainprofile DP 37 to 42” is understood as meaning an increase of the amountof amylopectin side chains with a DP of 37 to 42 by at least 5%,preferably by at least 10% and especially preferably by at least 15% incomparison with the amount of amylopectin side chains with a DP of 37 to42 from wild-type plants.

In connection with the present invention, the term modified “side chainprofile DP 62 to 123” is understood as meaning an increase of the amountof amylopectin side chains with a DP of 62 to 123 by at least 20%,preferably by at least 35%, especially preferably by at least 50% incomparison with the amount of amylopectin side chains with a DP of 62 to123 from wild-type plants.

The side chain profile is determined via determining the percentage of aspecific group of side chains in the total of all side chains in the GPCchromatogram. To this end, the total area under the line of the GPCchromatogram is divided into individual segments, each of whichrepresents groups of side chains of different lengths. The chosensegments contain side chains with the following degree of polymerization(DP=number of glucose monomers within one side chain): DP<11, DP12-18,DP19-24, DP25-30, DP31-36, DP37-42, DP43-48, DP49-55, DP56-61 andDP62-123. To elution volume with the molecular mass, the GPC column usedis calibrated with dextran standards (Fluka, Product# 31430). Thedextrans used, their associated molecular mass and the elution volumesare shown in FIG. 9. Using the resulting calibration graph, the elutiondiagram is shown as a molecular weight distribution. To determine themolecular weight of the individual side chains, a molecular weight of162 was set for glucose. The total area under the line in the GPCchromatogram is set as 100% and the percentage of the areas of theindividual segments is calculated based on the percentage of the totalarea.

In a further especially preferred embodiment, the amylopectin of starchaccording to the invention, from plant cells according to the inventionor plants according to the invention, shows an increased amount of theamylopectin side chains with a DP of greater than 123 in comparison withthe amount of side chains with a DP of greater than 123 from amylopectinof wild-type plants.

The plant cells according to the invention may be used for theregeneration of intact plants.

The plants obtainable by regeneration of the transgenic plant cellsaccording to the invention are likewise subject-matter of the presentinvention.

The plant cells according to the invention may belong to any plantspecies, i.e. to both monocotyledonous and dicotyledonous plants. Theyare preferably plant cells of agricultural useful plants, i.e. plantswhich are grown by man for the purposes of nutrition or for technical,in particular industrial, purposes. The invention preferably relates tofibre-forming plants (for example flax, hemp, cotton), oil-storingplants (for example oilseed rape, sunflower, soybean), sugar-storingplants (for example sugar beet, sugar cane, sugar millet) andprotein-storing plants (for example legumes).

In a further preferred embodiment, the invention relates to fodderplants, in particular to fodder grasses and forage grasses (alfalfa,clover and the like) and vegetable plants (for example tomato, lettuce,chicory).

In a further preferred embodiment, the invention relates to plant cellsfrom starch-storing plants (for example wheat, barley, oats, rye,potato, maize, rice, pea, cassava), with plant cells from potato beingespecially preferred.

A multiplicity of techniques are available for introducing DNA into aplant host cell. These techniques encompass the transformation of plantcells with T-DNA using Agrobacterium tumefaciens or Agrobacteriumrhizogenes as transformation agent, the fusion of protoplasts,injection, the electroporation of DNA, the introduction of the DNA bymeans of the biolistic approach, and other possibilities.

The use of the agrobacteria-mediated transformation of plant cells hasbeen studied intensively and described sufficiently in EP 120516;Hoekema, Ind.: The Binary Plant Vector System Offsetdrukkerij KantersB.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. PlantSci. 4, 1-46 and in An et al. EMBO J. 4, (1985), 277-287. As regards thetransformation of potato, see, for example, Rocha-Sosa et al., EMBO J.8, (1989), 29-33.).

The transformation of monocotyledonous plants by means of vectors basedon transformation with agrobacterium has also been described (Chan etal., Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6,(1994) 271-282; Deng et al, Science in China 33, (1990), 28-34; Wilminket al., Plant Cell Reports 11, (1992), 76-80; May et al.,Bic)/Technology 13, (1995), 486-492; Conner and Domisse, Int. J. PlantSci. 153 (1992), 550-555; Ritchie et al, Transgenic Res. 2, (1993),252-265). An alternative system for the transformation ofmonocotyledonous plants is the transformation by means of the biolisticapproach (Wan and Lemaux, Plant Physiol. 104, (1994), 37-48; Vasil etal., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol.Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79,(1990), 625-631), protoplast transformation, the electroporation ofpartially permeabilized cells, the introduction of DNA by means of glassfibres. In particular the transformation of maize has been describedrepeatedly in the literature (cf., for example, WO95/06128, EP0513849,EP0465875, EP0292435; Fromm et al., Biotechnology 8, (1990), 833-844;Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al.,Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80,(1990), 721-726).

The successful transformation of other cereal species has also beendescribed, for example for barley (Wan and Lemaux, see above; Ritala etal., see above; Krens et al., Nature 296, (1982), 72-74) and wheat(Nehra et al., Plant J. 5, (1994), 285-297). All of the abovementionedmethods are suitable for the purposes of the present invention.

Any promoter which is active in plant cells is generally suitable forthe expression of the foreign nucleic acid molecule(s). The promoter maybe chosen in such a way that expression in the plants according to theinvention takes place constitutively or only in a specific tissue, at aparticular point in time of plant development or at a point in timedetermined by external factors. As regards the plant, the promoter maybe homologous or heterologous.

In a further embodiment of the invention, at least one antisense RNA isexpressed in plant cells in order to reduce the activity of one or moreSSIII proteins and/or BEI proteins and/or BEII proteins.

The present invention therefore also relates to a plant cell accordingto the invention, wherein said foreign nucleic acid molecules areselected from the group consisting of

-   -   a) DNA molecules encoding at least one antisense RNA which        brings about a reduction of the expression of at least one        endogenous gene encoding SSIII proteins and/or BEI proteins        and/or BEII proteins;    -   b) DNA molecules which, via a cosuppression effect, lead to a        reduction of the expression of at least one endogenous gene        encoding SSIII protein(s) and/or BEI protein(s) and/or BEII        protein(s);    -   c) DNA molecules encoding at least one ribozyme which        specifically cleaves transcripts of at least one endogenous gene        encoding SSIII proteins and/or BEI proteins and/or BEII        proteins; and    -   d) Nucleic acid molecules introduced by means of in-vivo        mutagenesis which lead to a mutation or insertion of a        heterologous sequence in at least one endogenous gene encoding        SSIII protein(s) and/or BEI protein(s) and/or BEII protein(s),        the mutation or insertion bringing about a reduction of the        expression of at least one gene encoding SSIII protein(s) and/or        BEI protein(s) and/or BEII protein(s), or the synthesis of        inactive SSIII and/or BEI and/or BEII proteins;    -   e) DNA molecules which simultaneously encode at least one        antisense RNA and at least one sense RNA, where said antisense        RNA and said sense RNA form a double-stranded RNA molecule which        brings about a reduction of the expression of at least one        endogenous gene encoding SSIII protein(s) and/or BEI protein(s)        and/or BEII protein(s);    -   f) DNA molecules containing transposons, the integration of the        transposon sequences leading to a mutation or an insertion in at        least one endogenous gene encoding SSIII protein(s) and/or BEI        protein(s) and/or BEII protein(s) which brings about a reduction        of the expression of at least one gene encoding an SSIII        protein(s) and/or BEI protein(s) and/or BEII protein(s), or        which results in the synthesis of inactive SSIII and/or BEI        and/or BEII proteins; and    -   g) T-DNA molecules which, owing to the insertion into at least        one endogenous gene encoding SSIII protein(s) and/or BEI        protein(s) and/or BEII protein(s), brings about a reduction of        the expression of at least one endogenous gene encoding SSIII        protein(s) and/or BEI protein(s) and/or BEII protein(s), or        which result in the synthesis of inactive SSIII and/or BEI        and/or BEII proteins.

In a further aspect, the present invention relates to any kind ofpropagation material of plants according to the invention.

A further aspect of the present invention relates to the use of thenucleic acid molecules described herein for the generation of the plantcells and plants according to the invention.

A further aspect of the present invention relates to a compositioncomprising at least one of the above nucleic acid molecules, where theat least one nucleic acid molecule, after introduction into a plantcell, leads to the reduction of at least one SSIII protein which occursendogenously in the plant cell and at least one BEII protein whichoccurs endogenously in the plant cell and preferably furthermore to thereduction of at least one BEI protein which occurs endogenously in theplant cell. The composition may comprise one or more nucleic acidconstructs (cf. above).

A further aspect of the present invention relates to the use of thecompositions according to the invention for the generation of the plantcells and plants according to the invention, and to a host cell, inparticular a plant cell, containing the composition according to theinvention.

Yet a further aspect of the present invention relates to atransformation system in plant cells, comprising at least one nucleicacid molecule and at least one plant cell, where the at least onenucleic acid molecule leads to the reduction of the activity of in eachcase at least one of the SSIII, BEI and BEII proteins which occurendogenously in the plant cell unless the activity of these proteins hasbeen reduced already by an existing genetic modification of said plantcell. For the purposes of the present invention, “transformation system”thus relates to a combination of at least one plant cell to betransformed and at least one nucleic acid molecule as described abovewhich is used for the transformation. Further components with which theskilled worker in the field of the transformation of plant cells isfamiliar and which are required in the transformation process, includingbuffers and the like, may be present in the transformation systemaccording to the invention.

DESCRIPTION OF THE FIGURES

FIG. 1:

A graphic representation of the viscosity characteristics of starch frompotato plants. The analysis was carried out using a Rapid Visco Analyser(Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW2102, Australia). The conditions under which the analysis was carriedout are described under RVA analytical method 1 in the chapter “GeneralMethods”. The test starch was isolated from tubers of wild-type plants(WT), plants with a reduced activity of an SSIII protein and of a BEIprotein (038VL008 and 038VL107) or from plants with a reduced activityof an SSIII protein and a BEI protein and a BEII protein (110CF003 and108CF041). The starch was isolated by the method described under“Examples”, “Starch extraction process for potatoes”.

FIG. 2:

A graphic representation of the viscosity characteristics of starch frompotato plants. The analysis was carried out using a Rapid Visco Analyser(Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW2102, Australia). The conditions under which the analysis was carriedout are described under RVA analytical method 2 in the chapter “GeneralMethods”. The test starch was isolated from tubers of wild-type plants(WT), plants with a reduced activity of an SSIII protein and of a BEIprotein (038VL008 and 038VL107) or from plants with a reduced activityof an SSIII protein and a BEI protein and a BEII protein (110CF003 and108CF041). The starch was isolated by the method described under“Examples”, “Starch extraction process for potatoes”.

FIG. 3:

A graphic representation of the viscosity characteristics of starch frompotato plants. The analysis was carried out using a Rapid Visco Analyser(Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW2102, Australia). The conditions under which the analysis was carriedout are described under RVA analytical method 3 in the chapter “GeneralMethods”. The test starch was isolated from tubers of wild-type plants(WT), plants with a reduced activity of an SSIII protein and of a BEIprotein (038VL008 and 038VL107) or from plants with a reduced activityof an SSIII protein and a BEI protein and a BEII protein (110CF003 and108CF041). The starch was isolated by the method described under“Examples”, “Starch extraction process for potatoes”.

FIG. 4:

Scanning-electron micrograph of a potato starch granule isolated fromwild-type plants.

FIG. 5:

Scanning-electron micrograph of a potato starch granule isolated fromplants with a reduced activity of an SSIII protein and of a BEI proteinand of a BEII protein (110CF003).

FIG. 6:

Schematic representation of the vector pGSV71-α-BEII-basta, which wasused for the retransformation of plants in which a reduced activity ofan SSIII protein and of a BEI protein is already observed.

(RB, left T-DNA border, LB, right T-DNA border; CaMV35, cauliflowermosaic virus 35S promoter; NOS, polyadenylation sequence of theAgrobacterium tumefaciens nopaline synthase gene; OCS, polyadenylationsequence of the Agrobacterium tumefaciens octopine synthase gene; B33,promoter of the potato patatin gene; BEII, coding sequences of thepotato BEII gene; bar, sequence encoding a Streptomyces hygroscopicusphosphinothricin acetyltransferase).

FIG. 7:

Schematic representation of the vector pB33-α-BE-α-SSIII-Kan, which wasused for the generation of transgenic plants with a reduced activity ofan SSIII protein and a BEI protein (RB, left T-DNA border, LB, rightT-DNA border; nos5′, promoter of the Agrobacterium nopaline synthasegene; nptII, gene encoding the activity of a neomycinphosphotransferase; nos3, polyadenylation sequence of the Agrobacteriumtumefaciens nopaline synthase gene; OCS, polyadenylation sequence of theAgrobacterium tumefaciens octopine synthase gene; B33, promoter of thepotato patatin gene; BE, coding sequences of the potato BEI gene; SSIII,coding sequences of the potato SSIII gene).

FIG. 8

The figure shows the entire elution diagram of the amylopectin fromstarches of lines 038VL008, 108CF041 and wild type. As shown in thefigure, the amount of bigger side chains in line 108CF041 is markedlyhigher in contrast to the background 038VL008 and/or the correspondingwild type.

FIG. 9

Calibration curve and table with corresponding dextran standards

FIG. 10

The figure shows the entire elution diagram of the amylopectin fromstarches of lines 038VL008, 108CF041 and wild type. In contrast to FIG.8, the x axis does not show the elution volume, but the molecularweight. The elution diagram of FIG. 8 as a function of the molecularweight distribution is shown with the aid of the calibration graph ofFIG. 9.

FIG. 11

This represents the side chain profile distribution of the amylopectinfrom plants of line 038VL008 in comparison with the side chain profileof amylopectin from wild-type plants.

FIG. 12

This represents the side chain profile distribution of the amylopectinfrom plants of line 108CF041 in comparison with the side chain profileof amylopectin from wild-type plants.

DESCRIPTION OF THE SEQUENCES Seq ID 1:

Nucleic acid sequence of the potato (Solanum tuberosum) starch synthaseSSIII with indication of the sequences which encode the correspondingSSIII protein.

Seq ID 2:

Amino acid sequence of a potato SSIII protein.

Seq ID 3:

Amino acid sequence of the Pfam cbm25 binding domain of the potato SSIIIprotein (Solanum tuberosum).

Seq ID 4:

Coding nucleic acid sequence of the potato (Solanum tuberosum) branchingenzyme BEI.

Seq ID 5:

Amino acid sequence of the potato (Solanum tuberosum) branching enzymeBEI.

Seq ID 6:

Coding nucleic acid sequence of the potato (Solanum tuberosum) branchingenzyme BEII.

Seq ID 7:

Amino acid sequence of the potato (Solanum tuberosum) branching enzymeBEII.

Seq ID 8:

PCR-amplified nucleic acid sequence of the potato (Solanum tuberosum)branching enzyme BEII.

General Methods

The following methods were used in the examples:

Starch Analysis

a) Determination of the Amylose Content and of the Amylose/AmylopectinRatio

Starch was isolated from potato plants by standard methods, and theamylose content and the amylose:amylopectin ratio was determined by themethod described by Hovenkamp-Hermelink et al. (Potato Research 31,(1988), 241-246).

b) Determination of the Phosphate Content

In starch, the positions C2, C3 and C6 of the glucose units can bephosphorylated. To determine the C6-P content of starch, 50 mg of starchare hydrolysed for 4 h at 95° C. in 500 μl of 0.7 M HCl. The samples arethen centrifuged for 10 minutes at 15 500 g and the supernatants areremoved. 7 μl of the supernatants are mixed with 193 μl of imidazolebuffer (100 mM imidazole, pH 7.4; 5 mM MgCl₂, 1 mM EDTA and 0.4 mM NAD).The measurement was carried out in a photometer at 340 nm. After thebase absorption had been established, the enzyme reaction was started byaddition of 2 units glucose-6-phosphate dehydrogenase (from Leuconostocmesenteroides, Boehringer Mannheim). The change in absorption isdirectly proportional to the concentration of the G-6-P content of thestarch.

The total phosphate content was determined by the method of Ames(Methods in Enzymology VIII, (1966), 115-118).

Approximately 50 mg of starch are treated with 30 μl of ethanolicmagnesium nitrate solution and ashed for 3 hours at 500° C. in a muffleoven. The residue is treated with 300 μl of 0.5 M hydrochloric acid andincubated for 30 minutes at 60° C. One aliquot is subsequently made upto 300 μl 0.5 M hydrochloric acid and this is added to a mixture of 100μl of 10% ascorbic acid and 600 μl of 0.42% ammonium molybdate in 2 Msulphuric acid and incubated for 20 minutes at 45° C.

This is followed by a photometric determination at 820 nm with aphosphate calibration series as standard.

c) Determination of the Gel Strength (Texture Analyser)

1.5 g of starch (DM) are gelatinized in the RVA apparatus in 25 ml of anaqueous suspension (temperature programme: see item d) “Determination ofthe viscosity characteristics by means of a Rapid Visco Analyser (RVA)”)and subsequently stored for 24 hours at room temperature in a sealedcontainer. The samples are fixed under the probe (round piston withplanar surface) of a Texture Analyser TA-XT2 from Stable Micro Systems(Surrey, UK) and the gel strength was determined using the followingparameters:

Test speed 0.5 mm/s Depth of penetration 7 mm Contact surface 113 mm²Pressure 2 g

d) Determination of the Viscosity Characteristics by Means of a RapidVisco Analyser (RVA) Standard Method

2 g of starch (DM) are taken up in 25 ml of H₂O (VE-type water,conductivity of at least 15 mega ohm) and used for the analysis in aRapid Visco Analyser (Newport Scientific Pty Ltd., Investmet SupportGroup, Warriewod NSW 2102, Australia). The apparatus is operatedfollowing the manufacturer's instructions. The viscosity values areindicated in RVUs in accordance with the manufacturer's operatingmanual, which is incorporated into the description herewith byreference. To determine the viscosity of the aqueous starch solution,the starch suspension is first heated for one minute at 50° C. (step 1),and then heated from 50° C. to 95° C. at a rate of 12° C. per minute(step 2). The temperature is then held for 2.5 minutes at 95° C. (step3). Then, the solution is cooled from 95° C. to 50° C. at a rate of 12°C. per minute (step 4). The viscosity is determined during the entireduration.

In particular in those cases where the limits of the measuring range ofthe RVA were insufficient when 2.0 g (DM) of starch in 25 ml of H₂O(VE-type water, conductivity of at least 15 mega ohm) were weighed in,only 1.5 g of starch (DM) were taken up in 25 ml of H₂O (VE-type water,conductivity of at least 15 mega ohm).

For reasons of comparison with the prior art, a modified temperatureprofile was additionally used in some cases.

The following temperature profiles were used:

RVA Analytical Method 1:

To determine the viscosity of a 6% aqueous starch solution, the starchsuspension is first stirred for 10 seconds at 960 rpm and subsequentlyheated at 50° C. at a stirring speed of 160 rpm, initially for a minute(step 1). The temperature was then raised from 50° C. to 95° C. at aheating rate of 12° C. per minute (step 2). The temperature is held for2.5 minutes at 95° C. (step 3) and then cooled from 95° C. to 50° C. at12° C. per minute (step 4). In the last step (step 5), the temperatureof 50° C. is held for 2 minutes.

After the programme has ended, the stirrer is removed and the beakercovered. The gelatinized starch is now available for the textureanalysis after 24 hours.

RVA Analytical Method 2:

To determine the viscosity of a 6% aqueous starch solution, the starchsuspension is first stirred for 10 seconds at 960 rpm and subsequentlyheated at 50° C. at a stirring speed of 160 rpm, initially for twominutes (step 1). The temperature was then raised from 50° C. to 95° C.at a heating rate of 1.5° C. per minute (step 2). The temperature isheld for 15 minutes at 95° C. (step 3) and then cooled from 95° C. to50° C. at 1.5° C. per minute (step 4). In the last step (step 5), thetemperature of 50° C. is held for 15 minutes.

After the programme has ended, the stirrer is removed and the beakercovered. The gelatinized starch is now available for the textureanalysis after 24 hours.

RVA Analytical Method 3:

To determine the viscosity of a 10% aqueous starch solution, the starchsuspension is first stirred for 10 seconds at 960 rpm and subsequentlyheated at 50° C. at a stirring speed of 160 rpm, initially for twominutes (step 1). The temperature was then raised from 50° C. to 95° C.at a heating rate of 1.5° C. per minute (step 2). The temperature isheld for 15 minutes at 95° C. (step 3) and then cooled from 95° C. to50° C. at 1.5° C. per minute (step 4). In the last step (step 5), thetemperature of 50° C. is held for 15 minutes. This profile of the RVAanalysis corresponds to the one employed in WO 9634968.

After the programme has ended, the stirrer is removed and the beakercovered. The gelatinized starch is now available for the textureanalysis after 24 hours.

The profile of the RVA analysis contains parameters which are shown forthe comparison of different measurements and substances. In the contextof the present invention, the following terms are to be understood asfollows:

1. Maximum Viscosity (RVA Max)

The maximum viscosity is understood as meaning the highest viscosityvalue, measured in RVUs, obtained in step 2 or 3 of the temperatureprofile.

2. Minimum Viscosity (RVA Min)

The minimum viscosity is understood as meaning the lowest viscosityvalue, measured in RVUs, observed in the temperature profile after themaximum viscosity. Normally, this takes place in step 3 of thetemperature profile.

3. Final Viscosity (RVA Fin)

The final viscosity is understood as meaning the viscosity value,measured in RVUs, observed at the end of the measurement.

4. Setback (RVA Set)

What is known as the “setback” is calculated by subtracting the value ofthe final viscosity from that of the minimum occurring after the maximumviscosity in the curve.

5. Gelatinization Temperature (RVA T)

The gelatinization temperature is understood as meaning the point intime of the temperature profile where, for the first time, the viscosityincreases drastically for a brief period.

e) Analysis of the Side-Chain Distribution of the Amylopectin by Meansof Ion-Exchange Chromatography

To separate amylose and amylopectin, 200 mg of starch are dissolved in50 ml reaction vessels, using 12 ml of 90% (v/v) DMSO in H₂O. Afteraddition of 3 volumes of ethanol, the precipitate is separated bycentrifugation for 10 minutes at about 1800 g at room temperature (RT).The pellet is then washed with 30 ml of ethanol, dried and dissolved in40 ml of 1% (w/v) NaCl solution at 75° C. After the solution has cooledto 30° C., approximately 90 mg of thymol are added slowly, and thissolution is incubated for at least 60 h at 30° C. The solution is thencentrifuged for 30 minutes at 2000 g (RT). The supernatant is thentreated with 3 volumes of ethanol, and the amylopectin which settles outis separated by centrifugation for 5 minutes at 2000 g (RT).

The pellet (amylopectin) is then washed with ethanol and dried usingacetone. By addition of DMSO to the pellet, one obtains a 1% solution,of which 200 μl are treated with 345 μl of water, 10 μl of 0.5 M sodiumacetate (pH 3.5) and 5 μl of isoamylase (dilution 1:10; Megazyme) andincubated for about 16 hours at 37° C. A 1:5 aqueous dilution of thisdigest is subsequently filtered through a 0.2 μm filter, and 100 μl ofthe filtrate are analysed by ion chromatography (HPAEC-PAD, Dionex).Separation was performed using a PA-100 column (with suitableprecolumn), while detection was performed amperometrically. The elutionconditions were as follows:

Solution A—0.15M NaOH

Solution B—1 M sodium acetate in 0.15M NaOH

TABLE 1 Composition of the elution buffer for the side chain analysis ofthe amylopectin at different times during the HPEAC-PAD Dionex analysis.Between the times stated, the composition of the elution buffer changesin each case linearly. t (min) Solution A (%) Solution B (%)  5 0 100 3530 70 45 32 68 60 100 0 70 100 0 72 0 100 80 0 100 Stop

The determination of the relative amount of short side chains in thetotal of all side chains is carried out via the determination of thepercentage of a particular side chain in the total of all side chains.The total of all side chains is determined via the determination of thetotal area under the peaks which represent the polymerization degrees ofDP6 to 26 in the HPCL chromatogram.

The percentage of a particular side chain in the total of all sidechains is determined via the determination of the ratio of the areaunder the peak which represents this side chain in the HPLC chromatogramto the total area. The programme Chromelion 6.20 Version 6.20 fromDionex, USA, was used for determining the peak areas.

f) Granule Size Determination

Starch was extracted from potato tubers by standard methods (seeExamples). The granule size determination was then carried out using aphotosedimentometer of type “Lumosed FS1” from Retsch GmbH, Germany,using the software V.2.3. The software settings were as follows:

Substance data: Calibration No. 0 Density [kg/m³] 1500 Sedimentationfluid: Type Water Viscosity [Pa s] 0.001 Density [kg/m³] 1000 Addition —Recordings 5 min Cut-off [μm] 250 Passage [%] 100 Measuring range4.34-117.39 μm Calibration N Temperature 20° C.

The granule size distribution was determined in aqueous solution and wascarried out following the manufacturer's instructions and on the basisof the literature by, for example, H. Pitsch, Korngröβenbestimmung[granule size determination]; LABO-1988/3 Fachzeitschrift fürLabortechnik, Darmstadt.

g) Scanning Electron Micrographs (SEM)

To study the surface of the starch samples, the latter were dusted ontothe sample holder using a conductive adhesive. To avoid charging, thesample holders were finally sputtered with a 4 nm Pt coating. The starchsamples were studied using the field emission scanning electronmicroscope JSM 6330 F (Jeol) at an accelerating voltage of 5 kV.

h) Determination of the Activity of the SSIII, BEI and BEII Proteins

These were carried out as specified in the examples.

Examples Generation of the Expression Vector ME5/6

pGSV71 is a derivative of the plasmid pGSV7, which is derived from theintermediary vector pGSV1. pGSV1 is a derivative of pGSC1700, whoseconstruction has been described by Cornelissen and Vanderwiele (NucleicAcid Research 17, (1989), 19-25). pGSV1 was obtained from pGSC1700 bydeleting the carbenicillin resistance gene and deleting the T-DNAsequences of the TL-DNA region of the plasmid pTiB6S3.

pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar etal., Gene 2, (1977), 95-113) and the replication origin of thePseudomonas plasmid pVS1 (Itoh et al., Plasmid 11, (1984), 206). pGSV7additionally contains the selectable marker gene aadA, from theKlebsiella pneumoniae transposon Tn1331, which confers resistance to theantibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24 (3),(1990), 218-226; Tolmasky and Crosa, Plasmid 29(1), (1993), 31-40)

The plasmid pGSV71 was obtained by cloning a chimeric bar gene betweenthe border regions of pGSV7. The chimeric bar gene contains thecauliflower mosaic virus promoter sequence for transcriptionalinitiation (Odell et al., Nature 313, (1985), 180), the Streptomyceshygroscopicus bar gene (Thompson et al., Embo J. 6, (1987), 2519-2523)and the 3′-untranslated region of the pTiT37 T-DNA nopalin synthase genefor transcriptional termination and for polyadenylation. The bar geneconfers tolerance to the herbicide glufosinate-ammonium.

The T-DNA contains the right border sequence of the TL-DNA from theplasmid pTiB6S3 (Gielen et al., EMBO J. 3, (1984), 835-846) at position198-222. A polylinker sequence is located between nucleotide 223-249.The nucleotides 250-1634 contain the cauliflower mosaic virus p35S3promoter region (Odell et al., see above). The coding sequence of theStreptomyces hygroscopicus phosphinothricin resistance gene (bar)(Thompson et al. 1987, see above) is arranged between the nucleotides1635-2186. The two terminal codons at the 5′ end of the bar wild-typegene were replaced by the codons ATG and GAC. A polylinker sequence islocated between the nucleotides 2187-2205. The 260 by TaqI fragment ofthe untranslated 3′ end of the nopalin synthase gene (3′nos) from theT-DNA of the plasmid pTiT37 (Depicker et al., J. Mol. Appl. Genet. 1,(1982), 561-573) is located between the nucleotides 2206 and 2465. Thenucleotides 2466-2519 contain a polylinker sequence. The left bordersequence of the pTiB6S3 TL-DNA (Gielen et al., EMBO J. 3, (1984),835-846) is located between the nucleotides 2520-2544.

The vector pGSV71 was then cut using the enzyme PstI and madeblunt-ended. The B33 promoter and the ocs cassette was then excised fromthe vector pB33-Kan in the form of an EcoRI-HindIII fragment, madeblunt-ended and inserted into the vector pGSV71 which had been cut withPstI and made blunt-ended. The resulting vector was used as startingvector for the construction of ME5/6: An oligonucleotide containing thecleavage sites EcoRI, PacI, SpeI, SrfI, SpeI, NotI, PacI and EcoRI wasintroduced into the PstI cleavage site of the vector ME4/6 locatedbetween the B33 promoter and the ocs element, duplicating the PstIcleavage site. The resulting expression vector was termed ME5/6.

Description of the Vector pSK-Pac:

pSK-Pac is a derivative of pSK-Bluescript (Stratagene, USA) in which aPacI cleavage site was introduced at each flank of the multiple cloningsite (MCS).

Generation of Transgenic Potato Plants with a Reduced Gene Expression ofa BEI, SSIII and BEII Gene

To generate transgenic plants with a reduced activity of a BEI, an SSIIIand a BEII protein, transgenic plants with a reduced activity of a BEIand an SSIII protein were generated in a first step. To this end, theT-DNA of the plasmid pB33-αBEI-αSSIII-Kan was transferred into potatoplants with the aid of agrobacteria as described by Rocha-Sosa et al.(EMBO J. 8, (1989), 23-29).

To construct the plasmid pB33-αBEI-αSSIII-Kan (see FIG. 7), theexpression vector pBin33-Kan was constructed in a first step. To thisend, the promoter of the Solanum tuberosum patatin gene B33 (Rocha-Sosaet al., 1989, see above) was ligated in the form of a DraI fragment(nucleotides—1512-+14) into the SstI-cut vector pUC19 (Genbank Acc. No.M77789), whose ends have been made blunt-ended with the aid of T4 DNApolymerase. This gave rise to the plasmid pUC19-B33. The B33 promoterwas excised from this plasmid using EcoRI and SmaI and ligated into thesuitably cut vector pBinAR. This gave rise to the plant expressionvector pBin33-Kan. The plasmid pBinAR is a derivative of the vectorplasmid pBin19 (Bevan, Nucl. Acid Research 12, (1984), 8711-8721) andwas constructed by Höfgen and Willmitzer (Plant Sci. 66, (1990),221-230). A 1631 by HindII fragment which contains a partial cDNAencoding the potato BEI enzyme (Kossmann et al., 1991, Mol. & Gen.Genetics 230(1-2):39-44) was then made blunt-ended and introduced intothe vector pBin33, which had previously been cut with SmaI, in antisenseorientation with regard to the B33 promoter (promoter of the Solanumtuberosum patatin gene B33; Rocha-Sosa et al., 1989). The resultingplasmid was cut open using BamHI. A 1363 by BamHI fragment containing apartial cDNA encoding the potato SSIII enzyme (Abel et al., 1996,loc.cit.) was introduced into the cleavage site, again in antisenseorientation with regard to the B33 promoter.

After the transformation, various lines of transgenic potato plants inwhose tubers a markedly reduced activity of a BEI and SSIII protein wasobserved were identified. The plants resulting from this transformationwere termed 038VL.

To detect the activity of soluble starch synthases (SSIII) bynon-denaturing gel electrophoresis, tissue samples of potato tubers weredigested in 50 mM Tris-HCl pH 7.6, 2 mM DTT, 2.5 mM EDTA, 10% glyceroland 0.4 mM PMSF. The electrophoresis was carried out in a MiniProtean IIchamber (BioRAD). The monomer concentration of the gels, which had athickness of 1.5 mm, amounted to 7.5% (w/v), and 25 mM Tris-Glycin pH8.4 acted as the gel and the running buffers. Identical amounts ofprotein extract were applied and separated for 2 h at 10 mA for eachgel.

The activity gels were subsequently incubated in 50 mM Tricine-NaOH pH8.5, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 1 mM ADP-glucose,0.1% (w/v) amylopectin and 0.5 M sodium citrate. Glucans formed werestained with Lugol's solution.

The BEI activity was likewise detected with the aid of non-denaturinggel electrophoresis:

To isolate proteins from plants, the sample material was comminuted inliquid nitrogen using a pestle and mortar, taken up in extraction buffer(50 mM sodium citrate, pH 6.5; 1 mM EDTA, 4 mM DTT), centrifuged (10min, 14,000 g, 4° C.) and then employed directly in the proteinconcentration measurement following the method of Bradford. Then, 5 to20 μg of total protein extract (as required) were treated with 4×loading buffer (20% glycerol, 125 mM Tris HCl, pH 6.8) and applied to aBE activity gel. The running buffer (RB) was composed as follows:RB=30.2 g Tris-base, pH 8.0, 144 g glycine per 1 I H₂O.

After running of the gel had ended, each of the gels was incubatedovernight at 37° C. in 25 ml of “phosphorylase buffer” (25 ml 1M sodiumcitrate pH 7.0, 0.47 g glucose-1-phosphate, 12.5 mg AMP, 2.5 mgphosphorylase a/b from rabbit). The gels were stained using Lugol'ssolution.

More in-depth analyses demonstrated that isolated starches from lines038VL008 and 038VL107, in which both the BEI and the SSIII protein werereduced, showed the highest phosphate content of all independenttransformants studied.

Plants of these lines were subsequently transformed with the plasmidpGSV71-αBEII-basta as described by Rocha-Sosa et al. (EMBO J. 8, (1989),23-29).

Plasmid pGSV71-αBEII-basta was constructed by screening a tuber-specificpotato cDNA library with a DNA fragment amplified using RT-PCR (Primer:5′-gggggtgttggctttgacta (SEQ ID NO: 9) and 5′-cccttctcctcctaatccca (SEQID NO: 10); Stratagene ProSTAR™ HF Single-Tube RT-PCR system) with totalRNA from tubers as template, following standard methods. In this manner,an approximately 1250 by DNA fragment (SEQ ID No. 8) was isolated andthen subcloned into the EcoRV cleavage site of the cloning vectorpSK-Pac (see hereinabove) in the form of an EcoRV-SmaI fragment andsubsequently ligated into the expression vector ME5/6 in antisenseorientation relative to the promoter in the form of a PacI fragment.This gave rise to the plasmid pGSV71-□BEII-basta (see FIG. 6).

Tuber tissue samples of the independent transformants were obtained fromthe plants obtained by transformation with the plasmidpGSV71-αBEII-basta, which were referred to as 108CF and 110CF, and theiramylose content was determined (see Methods). The starches from theindependent lines whose tubers had the highest amylose content were usedfor a further analysis of the starch characteristics. To prove that inthese plants not only the activity of a BEI and SSIII protein isreduced, but also that the activity of a BEII protein is reduced,another analysis was carried out with the aid of non-denaturing gelelectrophoresis. The analysis was carried out following the same methodas already carried out above for the analysis of the reducing BEIactivity, with the exception that the non-denaturing polyacrylamide gelcontained 0.5% of maltodextrin (Beba, 15% strength maltodextrin solutionfor newborns, Nestle) in addition to the above-described composition.The dextrin addition made it possible to show the different activitiesof the BEI and BEII proteins after incubation of the gels in“phosphorylase buffer” (25 ml 1M sodium citrate pH 7.0, 0.47 gglucose-1-phosphate, 12.5 mg AMP, 2.5 mg phosphorylase a/b from rabbit)overnight at 37° C., followed by staining with Lugol's solution in agel.

Potato Starch Extraction Process

All tubers of one line (4 to 5 kg) are processed jointly in acommercially available juice extractor (Multipress automatic MP80,Braun). The starch-containing fruit water is collected in a 10-I bucket(ratio bucket height: bucket diameter=approx. 1.1) containing 200 ml oftap water together with a spoon-tipful (approx. 3-4 g) of sodiumdisulphite. The bucket is subsequently filled completely with tap water.After the starch has been allowed to settle for 2 hours, the supernatantis decanted off, the starch is resuspended in 10 l of tap water andpoured over a sieve with a mesh size of 125 μm. After 2 hours (starchhas again settled at the bottom of the bucket), the aqueous supernatantis again decanted off. This wash step is repeated 3 more times so thatthe starch is resuspended a total of 5 times in fresh tap water.Thereafter, the starches are dried at 37° C. to a water content of12-17% and homogenized using a pestle and mortar. The starches are nowavailable for analyses.

Example 2

Analysis of the Starch from Plants with Reduced BEI, SSIII and BEII GeneExpression

The starch from various independent lines of the transformations 108CFand 110CF described in Example 1 were isolated from potato tubers. Thephysico-chemical properties of this starch were subsequently analysed.The results of the characterization of the modified starches are shownin Table 2 (Tab. 2) for an example of a selection of certain plantlines. The analyses were carried out by the methods describedhereinabove.

Tables 2, 3 and 4 which follow summarize the results of the RVA analysisbased on starch from wild-type plants:

RVA Analytical Method 1

TABLE 2 Parameters of the RVA analysis of starch isolated from wild-typeplants (cv. Desiree), plants with a reduced activity of an SSIII and aBEI protein (038VL008, 038VL107), and of plants with a reduced activityof an SSIII and of a BEI and of a BEII protein (108CF041, 110CF003) inpercent based on data of starch of the wild type. RVA RVA RVA RVA RVAGel Max (%) Min (%) Fin (%) Set (%) T (%) strength cv. 100 100 100 100100 100 Desiree 038VL008 158.7 69.8 72.0 79.5 73.0 55.4 108CF041 59.689.9 227.5 693.7 150.2 532.3 038VL107 151.1 94.3 94.0 93.0 82.2 52.2110CF003 106.4 158.6 265.0 625.7 151.5 737.1 The RVA analysis wascarried out as described in Analytical method 1.

RVA Analytical Method 2

TABLE 3 Parameters of the RVA analysis of starch isolated from wild-typeplants (cv. Desiree), plants with a reduced activity of an SSIII and aBEI protein (038VL008, 038VL107), and of plants with a reduced activityof an SSIII and of a BEI and of a BEII protein (108CF041, 110CF003) inpercent based on data of starch of the wild type. RVA RVA RVA RVA RVAGel Max (%) Min (%) Fin (%) Set (%) T (%) strength cv. 100 100 100 100100 100 Desiree 038VL008 167.1 40.4 52.6 77.6 54.2 63.0 108CF041 44.582.5 187.5 402.7 137.4 412.2 038VL107 152.0 76.1 81.9 93.8 76.9 51.7110CF003 92.4 172.2 n.d. n.d. 139.0 795.0 The RVA analysis was carriedout as described in Analytical method 2.

RVA Analytical Method 3

TABLE 4 Parameters of the RVA analysis of starch isolated from wild-typeplants (cv. Desiree), plants with a reduced activity of an SSIII and aBEI protein (038VL008, 038VL107), and of plants with a reduced activityof an SSIII and of a BEI and of a BEII protein (108CF041, 110CF003) inpercent based on data of starch of the wild type. RVA RVA RVA RVA RVAGel Max (%) Min (%) Fin (%) Set (%) T (%) strength cv. 100 100 100 100100 100 Desiree 038VL008 n.d. 50.2 76.5 127.8 77.0 100.5 108CF041 74.7291.0 n.d. 205.7 236.0 630.3 038VL107 n.d. 84.5 86.4 90.1 102.3 58.1110CF003 89.8 259.7 n.d. n.d. 196.6 663.9 The RVA analysis was carriedout as described in Analytical method 3.

The following tables 5, 6 and 7 summarize the results of the RVAanalysis. The data do not refer to the wild type, but are the actualmeasurements:

RVA Analytical Method 1 (See Also FIG. 1)

TABLE 5 Parameters of the RVA analysis of starch isolated from wild-type plants (cv. Desiree), plants with a reduced activity of an SSIIIand a BEI protein (038VL008, 038VL107), and of plants with a reducedactivity of an SSIII and of a BEI and of a BEII protein (108CF041,110CF003) in RVUs. RVA RVA RVA RVA RVA Max Min Fin Set T Gel (RVU) (RVU)(RVU) (RVU) (RVU) strength cv. 255.05 162.33 210.25 47.92 4.6 25.1Desiree 038VL008 404.83 113.25 151.33 38.08 3.36 13.9 108CF041 152.08145.92 478.33 332.42 6.91 133.6 038VL107 385.5 153 197.58 44.58 3.7813.1 110CF003 271.5 257.42 557.25 299.83 6.97 185 The RVA analysis wascarried out as described in Analytical method 1.

RVA Analytical Method 2 (See Also FIG. 2)

TABLE 6 Parameters of the RVA analysis of starch isolated from wild-type plants (cv. Desiree), plants with a reduced activity of an SSIIIand a BEI protein (038VL008, 038VL107), and of plants with a reducedactivity of an SSIII and of a BEI and of a BEII protein (108CF041,110CF003) in RVUs. RVA RVA RVA RVA RVA Max Min Fin Set T Gel (RVU) (RVU)(RVU) (RVU) (RVU) strength cv. 212.17 113.75 169.25 55.5 28.78 23.8Desiree 038VL008 354.58 45.92 89 43.08 15.61 15 108CF041 94.33 93.83317.33 223.5 39.53 98.1 038VL107 322.58 86.58 138.67 52.08 22.13 12.3110CF003 196.08 195.92 n.d. n.d. 39.99 189.2 The RVA analysis wascarried out as described in Analytical method 2.

RVA Analytical Method 3 (See Also FIG. 3)

TABLE 7 Parameters of the RVA analysis of starch isolated from wild-type plants (cv. Desiree), plants with a reduced activity of an SSIIIand a BEI protein (038VL008, 038VL107), and of plants with a reducedactivity of an SSIII and of a BEI and of a BEII protein (108CF041,110CF003) in RVUs. RVA RVA RVA RVA RVA Max Min Fin Set T Gel (RVU) (RVU)(RVU) (RVU) (RVU) strength Desiree 819.67 207.67 314.25 106.58 16.8856.5 038VL008 n.d. 104.17 240.33 136.17 12.99 56.8 108CF041 612.33604.25 823.5 219.25 39.83 356.1 038VL107 n.d. 175.42 271.5 96.08 17.2732.8 110CF003 736.08 539.42 n.d. n.d. 33.18 375.1 The RVA analysis wascarried out as described in Analytical method 3.

Summary of the Phosphate and Amylose Analyses:

TABLE 8 Phosphate and amylose contents of starch isolated from wild-typeplants (cv. Desiree), plants with a reduced activity of an SSIII and aBEI protein (038VL008, 038VL107) and of plants with a reduced activityof an SSIII and of a BEI and of a BEII protein (108CF041, 110CF003).Phosphate Total Amylose Amylose No. Genotype in C6 (%) phosphate in (%)(%) (% WT) 1 cv. 100 100 22 100 Desiree 2 038VL008 346.4 255.2 19.4 85.83 108CF041 557.3 427.6 36.8 162.8 4 038VL107 225.5 182.8 19.7 87.2 5110CF003 446.4 348.3 34.6 153.1 The phosphate contents in the C6position of the glucose monomers and the total phosphate content of thestarch are indicated in percent based on starch from wild-type plants;amylose contents are indicated in percent amylose based on the totalamount of the starch, or in percent based on the amylose content ofstarch from wild-type plants.

The analysis of the side-chain distribution of the amylopectin wascarried out as described above. The table which follows is a summary ofthe contributions of the individual peak areas:

TABLE 9 The table shows a summary of the contributions of the individualpeak areas of the HPAEC chromatogram to the total peak area of wild-typeplants (cv. Desiree), of 038VL008 and 038Vl107 plants (potato plantswith reduced activity of a BEI protein and of an SSII protein) and ofselected lines of the transformations 108CF and 110CF (potato plantswith a reduced activity of an SSII protein and of a BEI protein and of aBEII protein). Glucose cv. units Desiree 038VL008 108CF041 038VL107110CF003 dp 6 1.52 4.16 1.88 2.39 0.86 dp 7 1.4 1.4 0.63 1.42 0.59 dp 81.23 0.77 0.33 0.99 0.38 dp 9 2.05 1.42 0.74 1.79 0.75 dp 10 3.55 2.81.74 3.33 1.77 dp 11 5.16 4.41 2.92 4.96 3.46 dp 12 6.25 5.77 4.47 6.225.17 dp 13 6.71 6.7 5.63 6.87 6.35 dp 14 6.75 7.06 6.35 6.99 7.38 dp 156.48 6.76 6.62 6.65 7.63 dp 16 6.07 5.99 6.34 6.11 7.13 dp 17 5.6 5.215.81 5.49 6.3 dp 18 5.28 4.78 5.87 5.11 5.98 dp 19 4.99 4.74 6.17 4.945.91 dp 20 4.76 4.65 6.07 4.78 5.64 dp 21 4.5 4.46 5.65 4.5 5.26 dp 224.16 4.12 5.07 4.2 4.7 dp 23 3.77 3.68 4.59 3.78 4.19 dp 24 3.44 3.364.24 3.42 3.75 dp 25 3.08 3.09 3.86 3.07 3.49 dp 26 2.73 2.8 3.36 2.773.03 dp 27 2.39 2.58 2.95 2.37 2.65 dp 28 2.07 2.26 2.39 2.01 2.1 dp 291.67 1.87 1.87 1.71 1.69 dp 30 1.38 1.58 1.54 1.35 1.3 dp 31 1.07 1.281.02 1.04 0.87 dp 32 0.79 0.96 0.7 0.75 0.6 dp 33 0.57 0.69 0.6 0.510.51 dp 34 0.36 0.43 0.39 0.32 0.34 dp 35 0.22 0.22 0.19 0.17 0.2 Total100 100 99.99 100.01 99.98 The number of glucose monomers in theindividual side chains is shown as dp.

-   -   The peak chain length, whose value is the mean of the two chain        lengths (given in DP) which contribute most to the total area        under the peaks of the HPAEC chromatogram, is—in the case of        debranched amylopectin—of wild-type plants at DP=13, in the case        of 038VL plants likewise at DP=13 and in the case of the 108CF        and 110CF plants, on average, at 15.    -   If the peak chain length of the transgenic plants is compared        with the peak chain length of amylopectin of wild-type plants,        the following values result for the peak chain length ratio (PCL        ratio):

PCL ratio for 038VL=13/13=1

PCL ratio for 108/110CF=15/13=1.15

-   -   In addition, the starch granule morphology was analysed using a        scanning electron microscope (SEM).    -   The surface of the starch granules of 108/110CF plants appears        coated or raised with pore formation.    -   Moreover, the granule size determination was carried out using a        “Lumosed”-type photosedimentometer from Retsch GmbH, Germany.    -   The mean granule size of untreated starch samples was determined        (Table 3).

TABLE 10 Mean granule size values of starch isolated from wild- typeplants (cv. Desiree), plants with a reduced activity of an SSIII and ofa BEI protein (038VL008, 038VL107), and of plants with a reducedactivity of an SSIII and of a BEI and of a BEII protein (108CF041,110CF003). Mean granule size [μm] Mean Sample granule size cv. Desiree29.7 038VL008 21.5 108CF041 20.8 038VL107 22.9 110CF003 20.7

Example 3 Analysis of the Amylopectin Side Chain Distribution by Meansof Gel Permeation Chromatography

To separate amylose and amylopectin, 100 mg of starch are dissolved in 6ml of 90% strength (v/v) DMSO with constant stirring. After addition of3 volumes of ethanol, the precipitate is separated off by centrifugationfor 10 minutes at 1 800 g at room temperature. The pellet issubsequently washed with 30 ml of ethanol, dried and dissolved in 10 mlof 1% strength (w/v) NaCl solution at 60° C. After cooling the solutionto 30° C., approximately 50 mg of thymol are added slowly, and thissolution is incubated for 2 to 3 days at 30° C. The solution issubsequently centrifuged for 30 minutes at 2 000 g at room temperature.The supernatant is treated with three volumes of ethanol, and theamylopectin which precipitates is separated off by centrifugation for 5minutes at 2 000 g at room temperature. The pellet (amylopectin) iswashed with 10 ml of 70% strength (v/v) ethanol, centrifuged for 10minutes at 2 000 g at room temperature and then dried using acetone.

10 mg of amylopectin are subsequently stirred for 10 minutes at 70° C.in 250 μl of 90% strength (v/v) DMSO. 375 μl of water at a temperatureof 80° C. are added to the solution until dissolution is complete.

200 μl of this solution are treated with 300 μl of a 16.6 mM sodiumacetate solution pH 3.5 and 2 μl of isoamylase (0.24 μ/μl, Megazyme,Sydney, Australia) and the mixture is incubated for 15 hours at 37° C.

A 1:4 dilution of this aqueous isoamylase reaction mixture with DMSO,comprising 90 mM sodium nitrate, is subsequently filtered through a 0.2μm filter, and 24 μl of the filtrate is analysed chromatographically.Separation was carried out with two columns connected in series, first aGram PSS3000 (Polymer Standards Service, with suitable precolumn),followed by a Gram PSS100. Detection was by means of refraction indexdetector (RI 71, Shodex). The column was equilibrated with DMSOcomprising 90 mM sodium nitrate. It was eluted with DMSO comprising 90mM sodium nitrate at a flow rate of 0.7 ml/min over a period of 1 hour.

To correlate the elution volume with the molecular mass, the column usedwas calibrated with dextran standards. The dextrans used, theirmolecular mass and the elution volumes are shown in FIG. 9. Using theresulting calibration graph, the elution diagram was shown as amolecular weight distribution (FIG. 10).

The chromatograms obtained were further evaluated using the programWingpc Version 6 from Polymer Standards Service GmbH, Mainz, Germany.

The total area under the line of the GPC chromatogram was divided intoindividual segments, each of which represent groups of side chains ofdifferent lengths. The chosen segments contained glucan chains with thefollowing degree of polymerization (DP=number of glucose monomers withinone side chain): DP<11, DP12-18, DP19-24, DP25-30, DP31-36, DP37-42,DP43-48, DP49-55, DP56-61 and DP62-123. To determine the molecularweight of the individual side chains, a molecular weight of 162 wasassumed for glucose. The total area under the line in the GPCchromatogram was then set as 100%, and the percentage of the areas ofthe individual segments was calculated based on the percentage of thetotal area. Results obtained from this analysis are shown in Table 11.

TABLE 11 Side chain profiles DP 12 to 18, DP 19 to 24, DP 25 to 30, DP31 to 36, DP 37 to 42, DP 43-48, DP 49 to 55, DP 56 to 61 and DP 62 to123 for amylopectin isolated from wild-type plants (cv. Desiree) andfrom plants with a reduced activity of an SSIII and of a BEI and of aBEII protein (108CF041). The percentages indicate the modification ofthe individual side chain profiles based on amylopectin isolated fromwild-type plants. Wild type 08CF041c DP ≧ 11 100% 40% DP12-18 100% 50%DP19-24 100% 69% DP25-30 100% 91% DP31-36 100% 111% DP37-42 100% 116%DP43-48 100% 110% DP49-55 100% 107% DP56-61 100% 109% DP62-123 100% 157%

1. Plant cell which is genetically modified, the genetic modificationleading to the reduction of the activity of one or more SSIII proteinsoccurring endogenously in the plant cell and to the reduction of theactivity of one or more BEI proteins which occur endogenously in theplant cell and to the reduction of the activity of one or more BEIIproteins which occur endogenously in the plant cell in comparison tocorresponding plant cells, of wild-type plants, which have not beengenetically modified.
 2. Plant cell according to claim 1, wherein thegenetic modification is the introduction of one or more foreign nucleicacid molecules whose presence and/or expression leads to the reductionof the activity of one or more SSIII and BEI and BEII proteins occurringin the plant cell in comparison with corresponding plant cells, ofwild-type plants, which have not been genetically modified. 3.Transgenic plant cells according to claim 2, wherein said foreignnucleic acid molecules are selected from the group consisting of a) DNAmolecules encoding at least one antisense RNA which brings about areduction of the expression of at least one endogenous gene encodingSSIII proteins and/or BEI proteins and/or BEII proteins; b) DNAmolecules which, via a cosuppression effect, lead to a reduction of theexpression of at least one endogenous gene encoding SSIII protein(s)and/or BEI protein(s) and/or BEII protein(s); c) DNA molecules encodingat least one ribozyme which specifically cleaves transcripts of at leastone endogenous gene encoding SSIII proteins and/or BEI proteins and/orBEII proteins; and d) Nucleic acid molecules introduced by means ofin-vivo mutagenesis which lead to a mutation or insertion of aheterologous sequence in at least one endogenous gene encoding SSIIIprotein(s) and/or BEI protein(s) and/or BEII protein(s), the mutation orinsertion bringing about a reduction of the expression of at least onegene encoding SSIII protein(s) and/or BEI protein(s) and/or BEIIprotein(s), or resulting in the synthesis of inactive SSIII and/or BEIand/or BEII proteins; e) DNA molecules which simultaneously encode atleast one antisense RNA and at least one sense RNA, where said antisenseRNA and said sense RNA form a double-stranded RNA molecule which bringsabout a reduction of the expression of at least one endogenous geneencoding SSIII protein(s) and/or BEI protein(s) and/or BEII protein(s);f) DNA molecules containing transposons, the integration of thetransposon sequences leading to a mutation or an insertion in at leastone endogenous gene encoding SSIII protein(s) and/or BEI protein(s)and/or BEII protein(s) which brings about a reduction of the expressionof at least one gene encoding an SSIII protein(s) and/or BEI protein(w)and/or BEII protein(s), or which results in the synthesis of inactiveSSIII and/or BEI and/or BEII proteins; and/or g) T-DNA molecules which,owing to the insertion into at least one endogenous gene encoding SSIIIprotein(s) and/or BEI protein(s) and/or BEII protein(s), bring about areduction of the expression of at least one gene encoding SSIIIprotein(s) and/or BEI protein(s) and/or BEII protein(s), or which resultin the synthesis of Inactive SSIII and/or BEI and/or BEII proteins. 4.Plant cell according to one of claims 1 to 3, which synthesizes amodified starch in comparison with a wild-type plant cell which has notbeen genetically modified.
 5. Plant cell according to claim 4, whereinthe modified starch is characterized in that a) it has an amylosecontent of at least 30%, b) it has an increased phosphate content incomparison with starch from corresponding wild-type plant cells whichhave not been genetically modified, and c) it has an increased finalviscosity in the RVA analysis in comparison with wild-type plant cellswhich have not been genetically modified.
 6. Plant cell according toclaim 4 or 5, wherein the modified starch is characterized in that ithas an increased gel strength in comparison with starch from wild-typeplant cells which have not been genetically modified.
 7. Plant cellaccording to one of claims 4 to 6, wherein the modified starch Ischaracterized in that it has a modified side chain distribution and/or amodified starch granule morphology in comparison with starch fromwild-type plant cells which have not been genetically modified.
 8. Plantcontaining plant cells according to one of claims 1 to
 7. 9. Method forgenerating a plant cell which synthesizes a modified starch, comprisingthe genetic modification of the plant cell, the genetic modificationleading to the reduction of the activity of one or more SSIII proteinswhich occur endogenously in the plant cell and to the reduction of theactivity of one or more BEI proteins which occur endogenously in theplant cell and to the reduction of the activity of one or more BEIIproteins which occur endogenously in the plant cell, in comparison withcorresponding plant cells, of wild-type plants, which have not beengenetically modified.
 10. Method for generating a plant cell accordingto claim 9 which synthesizes a modified starch, wherein the plant cellis genetically modified by the introduction of one or more foreignnudeic acid molecules whose presence and/or expression leads to thereduction of the activity of in each case at least one SSIII, BEI andBEII protein in comparison with corresponding wild-type plant cellswhich have not been genetically modified.
 11. Method according to claim9 or 10, wherein the modified starch is characterized in that a) It hasan amylose content of at least 30%, b) it has an increased phosphatecontent in comparison with starch from corresponding wild-type plantcells which have not been genetically modified, and c) it has anincreased final viscosity in the RVA analysis in comparison with starchfrom corresponding wild-type plant cells which have not been geneticallymodified.
 12. Method for generating a genetically modified plant, inwhich a) a plant cell according to one of claims 9 to 11 is generated;b) a plant is regenerated from, or using, the plant cell generated inaccordance with a); and, c) if appropriate, further plants are generatedfrom the plant generated in accordance with step b).
 13. Method forgenerating a transgenic plant according to claim 12 which synthesizes amodified starch, in which a) a plant cell is genetically modified by theintroduction of one or more foreign nucleic acid molecules whosepresence and/or expression leads to the reduction of the activity of ineach case at least one SSIII, BEI and BEII protein in comparison withcorresponding wild-type plant cells which have not been geneticallymodified; b) a plant is regenerated from, or using, the cell generatedin accordance with a); and c) if appropriate, further plants aregenerated from the plants generated in accordance with step b). 14.Method according to claim 12 or 13, wherein the modified starch ischaracterized in that a) it has an amylose content of at least 30%, b)it has an increased phosphate content in comparison with starch fromcorresponding wild-type plant cells which have not been geneticallymodified, and c) it has an increased final viscosity in the RVA analysisin comparison with starch from corresponding wild-type plants which havenot been genetically modified.
 15. Plant according to claim 8 orobtainable by the method according to one of claims 12 to 14, which is astarch-storing plant.
 16. Plant according to claim 15, which is a potatoplant.
 17. Propagation material of plants according to one of claims 8or 15 to 16 containing at least one plant cell according to one ofclaims 1 to
 7. 18. Use of one or more nucleic acid molecules whichencode proteins with the enzymatic activity of at least one SSIII, atleast one BEI and/or at least one BEII protein or their fragments forthe generation of plant cells according to one of claims 1 to 7 or ofplants according to one of claims 8 or 15 to
 16. 19. Use of one or morenucleic acid molecules for the generation of plant cells according toone of claims 1 to 7 or of plants according to one of claims 8 or 15 to16, wherein the foreign nucleic acid molecule(s) are selected from thegroup consisting of: a) DNA molecules encoding at least one antisenseRNA which brings about a reduction of the expression of at least oneendogenous gene encoding SSIII protein(s) and/or BEI protein(s) and/orBEII protein(s); b) DNA molecules which, via a cosuppression effect,lead to a reduction of the expression of at least one endogenous geneencoding SSIII protein(s) and/or BEI protein(s) and/or BEII protein(s);c) DNA molecules encoding at least one ribozyme which specificallycleaves transcripts of at least one endogenous gene encoding SSIIIprotein(s) and/or BEI protein(s) and/or BEII protein(s); d) Nucleic acidmolecules introduced by means of in-vivo mutagenesis which lead to amutation or insertion of a heterologous sequence in at least oneendogenous gene encoding SSIII protein(s) and/or BEI protein(s) and/orBEII protein(s), the mutation or insertion bringing about a reduction ofthe expression of at least one gene encoding SSIII protein(s) and/or BEIprotein(s) and/or BEII protein(s), or resulting in the synthesis ofinactive SSIII and/or BEI and/or BEII proteins; e) DNA molecules whichsimultaneously encode at least one antisense RNA and at least one senseRNA, where said antisense RNA and said sense RNA form a double-strandedRNA molecule which brings about a reduction of the expression of atleast one endogenous gene encoding SSIII proteins and/or BEI proteinsand/or BEII proteins; f) DNA molecules containing transposons, theintegration of the transposon sequences leading to a mutation or aninsertion in at least one endogenous gene encoding SSIII protein(s)and/or BEI protein(s) and/or BEII protein(s) which brings about areduction of the expression of at least one gene encoding an SSIIIprotein(s) and/or BEI protein(s) and/or BEII protein(s), or which resultin the synthesis of inactive SSIII and/or BEI and/or BEII proteins; andg) T-DNA molecules which, owing to the insertion into at least oneendogenous gene encoding SSIII proteins and/or BEI proteins and/or BEIIproteins, bring about a reduction of the expression of at least one geneencoding SSIII protein(s) and/or BEI protein(s) and/or BEII protein(s),or which result in the synthesis of inactive SSIII and/or BEI and/orBEII proteins.
 20. Composition comprising at least one of the nucleicacid molecules defined in claim 19, where the at least one nucleic acidmolecule, after introduction into a plant cell, leads to the reductionof at least one SSIII protein which occurs endogenously in the plantcell and at least one BEII protein which occurs endogenously in theplant cell and preferably furthermore to the reduction of at least oneBEI protein which occurs endogenously in the plant cell.
 21. Compositionaccording to claim 20, characterized in that the presence of the atleast one nucleic acid molecule in said plant cells leads to a reductionof the activity of in each case at least one SSIII and BEI and BEIIprotein in comparison with corresponding wild-type plant cells whichhave not been genetically modified.
 22. Composition according to one ofclaims 20 to 21, the nucleic acid molecules being present in arecombinant nucleic acid molecule.
 23. Use of a composition according toone of claims 20 to 22, or containing at least one of the nucleic acidmolecules defined in claim 19, for generating a plant cell with areduced activity of one or more SSIII proteins which occur endogenouslyin the plant cell and a reduced activity of one or more BEI proteinswhich occur endogenously in the plant cell and a reduced activity of oneor more BEII proteins which occur endogenously in the plant cell, Incomparison with corresponding plant cells, of wild-type plants, whichhave not been genetically modified.
 24. Transformation system for plantcells containing at least one nucleic acid molecule and at least oneplant cell, the at least one nucleic acid molecule leading to thereduction of the activity of in each case at least one of the SSIII, BEIand BEII proteins which occur endogenously in the plant cell unless theactivity of these proteins has been reduced already by an existinggenetic modification of said plant cell.
 25. Host cell containing acomposition according to one of claims 20 to
 22. 26. Host cell accordingto claim 25, which is a transgenic plant cell containing the compositionaccording to one of claims 20 to
 22. 27. Starch which can be obtainedfrom plant cells according to one of claim 1 to 7 or 26 or from a plantaccording to one of claims 8 or 15 to 16 or from propagation materialaccording to claim
 17. 28. Starch according to claim 27, characterizedin that a) it contains amylose content of at least 30%, b) it has anincreased phosphate content in comparison with starch from correspondingwild-type plant cells which have not been genetically modified, and c)it has an increased final viscosity in the RVA analysis in comparisonwith starch from corresponding wild-type plants which have not beengenetically modified.
 29. Starch according to claim 27 or 28,characterized in that it has a modified side chain distribution incomparison with starch from corresponding wild-type plant cells whichhave not been genetically modified.
 30. Starch according to one ofclaims 27 to 29, characterized in that the granule morphology and/or themean granule size of the starch granules are modified in comparison withwild-type plant cells which have not been genetically modified. 31.Starch according to one of claims 27 to 30, which is a potato starch.32. Method for producing a starch according to one of claims 27 to 31,comprising the extraction of the starch from a plant according to one ofclaims 8 or 15 to 16 and/or from starch-storing parts of such a plantand/or from a plant cell according to one of claim 1 to 7 or 26 and/orfrom propagation material according to claim
 17. 33. Starch according toone of claims 27 to 31, obtainable by a method according to claim 32.34. Starch according to one of claim 27 to 31 or 33, characterized inthat it has at least one of the following characteristics: a) a finalviscosity in the RVA analysis of 6% (w/w) aqueous starch suspension ofat least 300 RVU, preferably at least 400 RVU, in particular at least500 RVU; b) a phosphate content in the C6 position of the glucosemonomers of the starch of 40 to 120 nmol, preferably 60 to 100 nmol, inparticular 80 to 100 nmol of C6-P per mg of starch; c) a porous surfaceof the starch granules in native form.
 35. Processing product, inparticular foodstuffs or feedstuffs, colour, adhesive, building orinsulating material, containing a starch according to one of claim 27 to31; 33 or
 34. 36. Starch-containing part of a plant according to one ofclaim 8, 15 or
 16. 37. Method for modifying the starch of a plant,comprising the method for generating a plant according to one of claims12 to 14 and obtaining starch from the plant or starch-containing partsthereof.
 38. Method according to claim 37, characterized in that themodification of the starch comprises: a) increasing the amylose contentof the starch; b) increasing the phosphate content of the starch, inparticular to a phosphate content in the C6-position of the glucosemonomers of the starch of 40 to 120 nmol, preferably 60 to 100 nmol, inparticular 80 to 100 nmol of C6-P per mg of starch; c) increasing thefinal viscosity of the starch, in particular to a final viscosity in theRVA analysis with 6% (w/w) aqueous starch suspension of at least 300RVU, preferably at least 400 RVU, in particular at least 500 RVU; d)increasing the gel strength of the gelatinized starch, and/or e) themorphology, in particular the surface structure, porosity and/or granulesize distribution of the native starch granules.